BioChemistry Midterm

58 Water
SUMMARY 2.1 Weak Interactions in Aqueous Systems discussion of the ionization of water and of weak acids
and bases dissolved in water.
The very different electronegativities of H and O
make water a highly polar molecule, capable of
forming hydrogen bonds with itself and with solutes. Pure Water Is Slightly Ionized
Hydrogen bonds are fleeting, primarily electrostatic, Water molecules have a slight tendency to undergo
and weaker than covalent bonds. Water is a good reversible ionization to yield a hydrogen ion (a proton)
solvent for polar (hydrophilic) solutes, with which it and a hydroxide ion, giving the equilibrium
forms hydrogen bonds, and for charged solutes, with
H2O ∆ H1 1 OH2 (2–1)
which it interacts electrostatically.
Nonpolar (hydrophobic) compounds dissolve Although we commonly show the dissociation product
poorly in water; they cannot hydrogen-bond with of water as H1, free protons do not exist in solution;
the solvent, and their presence forces an hydrogen ions formed in water are immediately hydrat-
energetically unfavorable ordering of water ed to form hydronium ions (H3O1). Hydrogen bonding
molecules at their hydrophobic surfaces. To between water molecules makes the hydration of dis-
minimize the surface exposed to water, nonpolar sociating protons virtually instantaneous:
compounds such as lipids form aggregates ÷
H O H O H O⫹ H ⫹ OH⫺
(micelles) in which the hydrophobic moieties are H H H
sequestered in the interior, associating through
hydrophobic interactions, and only the more polar The ionization of water can be measured by its elec-
moieties interact with water. trical conductivity; pure water carries electrical current
Weak, noncovalent interactions, in large numbers, as H3O1 migrates toward the cathode and OH2 toward
decisively influence the folding of macromolecules the anode. The movement of hydronium and hydroxide
such as proteins and nucleic acids. The most stable ions in the electric field is extremely fast compared with
macromolecular conformations are those in which that of other ions such as Na1, K1, and Cl2. This high
hydrogen bonding is maximized within the molecule ionic mobility results from the kind of “proton hopping”
and between the molecule and the solvent, and in shown in Figure 2–14. No individual proton moves very
which hydrophobic moieties cluster in the interior
of the molecule away from the aqueous solvent. Hydronium ion gives up a proton
The physical properties of aqueous solutions are H H
strongly influenced by the concentrations of solutes. O+ Proton hop
When two aqueous compartments are separated by
a semipermeable membrane (such as the plasma H
O
membrane separating a cell from its surroundings), H
water moves across that membrane to equalize the H O H
osmolarity in the two compartments. This tendency H H
O
for water to move across a semipermeable H
membrane produces the osmotic pressure.
H O
2.2 Ionization of Water, Weak Acids, H
and Weak Bases O H

H
Although many of the solvent properties of water can be H
explained in terms of the uncharged H2O molecule, the O
small degree of ionization of water to hydrogen ions H
(H1) and hydroxide ions (OH2) must also be taken into H
O
account. Like all reversible reactions, the ionization of
water can be described by an equilibrium constant. H
When weak acids are dissolved in water, they contribute Water accepts proton and
H1 by ionizing; weak bases consume H1 by becoming becomes a hydronium ion
protonated. These processes are also governed by equi- FIGURE 2–14 Proton hopping. Short “hops” of protons between a series
librium constants. The total hydrogen ion concentration of hydrogen-bonded water molecules result in an extremely rapid net
from all sources is experimentally measurable and is movement of a proton over a long distance. As a hydronium ion (upper
expressed as the pH of the solution. To predict the state left) gives up a proton, a water molecule some distance away (bottom)
of ionization of solutes in water, we must take into acquires one, becoming a hydronium ion. Proton hopping is much faster
account the relevant equilibrium constants for each than true diffusion and explains the remarkably high ionic mobility of H1
ionization reaction. We therefore turn now to a brief ions compared with other monovalent cations such as Na1 and K1.
2.2 Ionization of Water, Weak Acids, and Weak Bases 59
far through the bulk solution, but a series of proton In pure water at 25 8C, the concentration of water is
hops between hydrogen-bonded water molecules causes 55.5 M—grams of H2O in 1 L divided by its gram molecu-
the net movement of a proton over a long distance in a lar weight: (1,000 g/L)/(18.015 g/mol)—and is essen-
remarkably short time. (OH2 also moves rapidly by pro- tially constant in relation to the very low concentrations
ton hopping, but in the opposite direction.) As a result of H1 and OH2, namely 1 3 1027 M. Accordingly, we can
of the high ionic mobility of H1, acid-base reactions in substitute 55.5 M in the equilibrium constant expression
aqueous solutions are exceptionally fast. As noted (Eqn 2–3) to yield
above, proton hopping very likely also plays a role in
biological proton-transfer reactions (Fig. 2–10; see also [H 1 ][OH 2 ]
Keq 5
Fig. 19–69b). [55.5 M]
Because reversible ionization is crucial to the role of
On rearranging, this becomes
water in cellular function, we must have a means of
expressing the extent of ionization of water in quantita- (55.5 M )(Keq ) 5 [H1][OH2] 5 Kw (2–4)
tive terms. A brief review of some properties of revers-
ible chemical reactions shows how this can be done. where Kw designates the product (55.5 M)(Keq), the ion
The position of equilibrium of any chemical reaction product of water at 25 8C.
is given by its equilibrium constant, Keq (sometimes The value for Keq, determined by electrical-
expressed simply as K). For the generalized reaction conductivity measurements of pure water, is 1.8 3 10216 M
at 25 8C. Substituting this value for Keq in Equation 2–4
A1B ∆ C1D (2–2)
gives the value of the ion product of water:
the equilibrium constant Keq can be defined in terms of
the concentrations of reactants (A and B) and products Kw 5 [H1][OH2] 5 (55.5 M )(1.8 3 10216 M )
(C and D) at equilibrium: 5 1.0 3 10214 M2
[C]eq[D]eq
Keq 5 Thus the product [H1][OH2] in aqueous solutions at
[A]eq[B]eq 25 8C always equals 1 3 10214 M2. When there are exactly
Strictly speaking, the concentration terms should be equal concentrations of H1 and OH2, as in pure water,
the activities, or effective concentrations in nonideal the solution is said to be at neutral pH. At this pH, the
solutions, of each species. Except in very accurate concentration of H1 and OH2 can be calculated from
work, however, the equilibrium constant may be the ion product of water as follows:
approximated by measuring the concentrations at
Kw 5 [H1][OH2] 5 [H1]2 5 [OH2]2
equilibrium. For reasons beyond the scope of this dis-
cussion, equilibrium constants are dimensionless. Solving for [H1] gives
Nonetheless, we have generally retained the concentra-
tion units (M) in the equilibrium expressions used in [H1] 5 2Kw 5 21 3 10214 M2
this book to remind you that molarity is the unit of [H1] 5 [OH2] 5 1027 M
concentration used in calculating Keq.
The equilibrium constant is fixed and characteristic As the ion product of water is constant, whenever [H1]
for any given chemical reaction at a specified tempera- is greater than 1 3 1027 M, [OH2] must be less than 1 3
ture. It defines the composition of the final equilibrium 1027 M, and vice versa. When [H1] is very high, as in a
mixture, regardless of the starting amounts of reactants solution of hydrochloric acid, [OH2] must be very low.
and products. Conversely, we can calculate the equilib- From the ion product of water we can calculate [H1] if
rium constant for a given reaction at a given tempera- we know [OH2], and vice versa.
ture if the equilibrium concentrations of all its reactants
and products are known. As we showed in Chapter 1
(p. 26), the standard free-energy change (DG8) is WORKED EXAMPLE 2–3 Calculation of [H1]
directly related to ln Keq. What is the concentration of H1 in a solution of 0.1 M
NaOH?
The Ionization of Water Is Expressed
by an Equilibrium Constant Solution: We begin with the equation for the ion product
of water:
The degree of ionization of water at equilibrium (Eqn 2–1)
is small; at 25 8C only about two of every 109 molecules Kw 5 [H1][OH2]
in pure water are ionized at any instant. The equilibrium With [OH2] 5 0.1 M, solving for [H1] gives
constant for the reversible ionization of water is
1 3 10214 M2 10214 M2
[H1 4 5
Kw
[H1][OH2] 2 5 5
Keq 5 (2–3) [OH ] 0.1 M 1021 M
[H2O]
5 10 213
M
60 Water
tration of hydrogen ions is 1.0 3 1027 M, the pH can be

WORKED EXAMPLE 2–4 Calculation of [OH2] calculated as follows:
What is the concentration of OH2 in a solution with an
1
H1 concentration of 1.3 3 1024 M? pH 5 log 5 7.0
1.0 3 1027
Solution: We begin with the equation for the ion product Note that the concentration of H1 must be expressed in
of water: molar (M) terms.
Kw 5 [H1][OH2] The value of 7 for the pH of a precisely neutral solu-
tion is not an arbitrarily chosen figure; it is derived from
With [H1] 5 1.3 3 1024 M, solving for [OH2] gives the absolute value of the ion product of water at 25 8C,
1 3 10214 M2 10214 M2 which by convenient coincidence is a round number.
[OH2 4 5
Kw
5 5 Solutions having a pH greater than 7 are alkaline or
[H ] 1
0.00013 M 1.3 3 1024 M
5 7.7 3 10 211
M basic; the concentration of OH2 is greater than that of
H1. Conversely, solutions having a pH less than 7 are
In all calculations be sure to round your answer to the acidic.
correct number of significant figures, as here. Keep in mind that the pH scale is logarithmic, not
arithmetic. To say that two solutions differ in pH by 1 pH
unit means that one solution has ten times the H1
The pH Scale Designates the H1 and concentration of the other, but it does not tell us the
OH2 Concentrations absolute magnitude of the difference. Figure 2–15
gives the pH values of some common aqueous fluids. A
The ion product of water, Kw, is the basis for the pH
cola drink (pH 3.0) or red wine (pH 3.7) has an H1 con-
scale (Table 2–6). It is a convenient means of designat-
centration approximately 10,000 times that of blood
ing the concentration of H1 (and thus of OH2) in any
(pH 7.4).
aqueous solution in the range between 1.0 M H1 and
The pH of an aqueous solution can be approximately
1.0 M OH2. The term pH is defined by the expression
measured with various indicator dyes, including litmus,
1 phenolphthalein, and phenol red. These dyes undergo
pH 5 log 5 2log [H1]
[H1] color changes as a proton dissociates from the dye
The symbol p denotes “negative logarithm of.” For a
precisely neutral solution at 25 8C, in which the concen- 0 1 m HCl
1
Gastric juice
TABLE 2–6 The pH Scale 2 Lemon juice
[H1] (M) pH [OH2] (M) pOH* 3 Cola, vinegar

0 Increasingly
10 (1) 0 10 214
14 acidic
Red wine
4
1021 1 10213 13 Beer
1022 2 10212 12 5 Black cofee
1023 3 10211 11 6
1024 4 10210 10 Milk, saliva
1025 5 1029 9 7 Neutral Human blood, tears
1026 6 1028 8 8
Seawater, egg white
1027 7 1027 7
1028 8 1026 6 9 Solution of baking
soda (NaHCO3)
1029 9 1025 5 10
10210 10 1024 4 Increasingly
11 basic
10211 11 1023 3
10212 12 1022 2 12 Household ammonia
10213 13 1021 1 Household bleach
13
10214 14 100 (1) 0
*The expression pOH is sometimes used to describe the basicity, or OH2 concentration, of a 14 1 m NaOH
solution; pOH is defined by the expression pOH 5 2log[OH2], which is analogous to the
expression for pH. Note that in all cases, pH 1 pOH 5 14. FIGURE 2–15 The pH of some aqueous fluids.
2.2 Ionization of Water, Weak Acids, and Weak Bases 61
molecule. Accurate determinations of pH in the chemi- completely ionized. Of more interest to biochemists is
cal or clinical laboratory are made with a glass electrode the behavior of weak acids and bases—those not com-
that is selectively sensitive to H1 concentration but pletely ionized when dissolved in water. These are
insensitive to Na1, K1, and other cations. In a pH meter, ubiquitous in biological systems and play important
the signal from the glass electrode placed in a test solu- roles in metabolism and its regulation. The behavior of
tion is amplified and compared with the signal generat- aqueous solutions of weak acids and bases is best
ed by a solution of accurately known pH. understood if we first define some terms.
Measurement of pH is one of the most important Acids may be defined as proton donors and bases as
and frequently used procedures in biochemistry. proton acceptors. When a proton donor such as acetic
The pH affects the structure and activity of biological acid (CH3COOH) loses a proton, it becomes the corre-
macromolecules; for example, the catalytic activity of sponding proton acceptor, in this case the acetate anion
enzymes is strongly dependent on pH (see Fig. 2–22). (CH3COO2). A proton donor and its corresponding pro-
Measurements of the pH of blood and urine are com- ton acceptor make up a conjugate acid-base pair
monly used in medical diagnoses. The pH of the blood (Fig. 2–16), related by the reversible reaction
plasma of people with severe, uncontrolled diabetes, for
CH3COOH ∆ CH3COO2 1 H1
example, is often below the normal value of 7.4; this
condition is called acidosis (described in more detail Each acid has a characteristic tendency to lose its
below). In certain other diseases the pH of the blood is proton in an aqueous solution. The stronger the acid,
higher than normal, a condition known as alkalosis. the greater its tendency to lose its proton. The tendency
Extreme acidosis or alkalosis can be life-threatening. ■ of any acid (HA) to lose a proton and form its conjugate
base (A2) is defined by the equilibrium constant (Keq)
for the reversible reaction
Weak Acids and Bases Have Characteristic Acid
HA ∆ H1 1 A2
Dissociation Constants
Hydrochloric, sulfuric, and nitric acids, commonly called for which
strong acids, are completely ionized in dilute aqueous [H1][A2]
Keq 5 5 Ka
solutions; the strong bases NaOH and KOH are also [HA]
Monoprotic acids O O
Acetic acid CH3C CH3C ⫹ H⫹
(Ka ⫽ 1.74 ⫻ 10⫺5 M) OH O⫺
pKa ⫽ 4.76
Ammonium ion NH⫹
4 NH3 ⫹ H⫹
(Ka ⫽ 5.62 ⫻ 10⫺10 M) pKa ⫽ 9.25
Diprotic acids
Carbonic acid
(Ka ⫽ 1.70 ⫻ 10⫺4 M); H2CO3 HCO⫺
3 ⫹ H
⫹
HCO⫺
3 CO32⫺ ⫹ H⫹
Bicarbonate pKa ⫽ 3.77* pKa ⫽ 10.2
(Ka ⫽ 6.31 ⫻ 10⫺11 M)
Glycine, carboxyl NH⫹

3 O NH⫹
3 O NH⫹
3 O NH2 O
(Ka ⫽ 4.57 ⫻ 10⫺3 M);
CH2C CH2C ⫹ H⫹ CH2C CH2C ⫹ H⫹
Glycine, amino
(Ka ⫽ 2.51 ⫻ 10⫺10 M) OH O⫺ O⫺ O⫺
pKa ⫽ 2.34 pKa ⫽ 9.60
Triprotic acids
Phosphoric acid
(Ka ⫽ 7.25 ⫻ 10⫺3 M);
Dihydrogen phosphate H3PO4 H2PO⫺
4 ⫹ H
⫹
H2PO⫺
4 HPO42⫺ ⫹ H⫹ HPO2⫺
4 PO3⫺
4 ⫹ H
⫹
(Ka ⫽ 1.38 ⫻ 10⫺7 M);
pKa ⫽ 2.14 pKa ⫽ 6.86 pKa ⫽ 12.4
Monohydrogen phosphate
(Ka ⫽ 3.98 ⫻ 10⫺13 M)
1 2 3 4 5 6 7 8 9 10 11 12 13
pH
FIGURE 2–16 Conjugate acid-base pairs consist of a proton donor and reactions for each pair are shown where they occur along a pH gradient.
a proton acceptor. Some compounds, such as acetic acid and ammonium The equilibrium or dissociation constant (Ka) and its negative logarithm,
ion, are monoprotic; they can give up only one proton. Others are diprotic the pKa, are shown for each reaction. *For an explanation of apparent dis-
(carbonic acid and glycine) or triprotic (phosphoric acid). The dissociation crepancies in pKa values for carbonic acid (H2CO3), see p. 67.
62 Water
Equilibrium constants for ionization reactions are usu- 9

ally called ionization constants or acid dissociation
8 CH3COO⫺
constants, often designated Ka. The dissociation con-
stants of some acids are given in Figure 2–16. Stronger 7 [CH3COOH] ⫽ [CH3COO⫺]
acids, such as phosphoric and carbonic acids, have
larger ionization constants; weaker acids, such as mono- 6
pH 5.76
hydrogen phosphate (HPO22 4 ), have smaller ionization
constants. 5 Buffering
pH region
Also included in Figure 2–16 are values of pKa,
4
which is analogous to pH and is defined by the equation pH 3.76
pH ⫽ pKa ⫽ 4.76
3
1
pKa 5 log 5 2log Ka
Ka
2 CH3COOH
The stronger the tendency to dissociate a proton, the
1
stronger is the acid and the lower its pKa. As we shall
now see, the pKa of any weak acid can be determined 0
quite easily. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
OH⫺ added (equivalents)
Titration Curves Reveal the pKa of Weak Acids 0 50 100%

Percent titrated
Titration is used to determine the amount of an acid in
a given solution. A measured volume of the acid is FIGURE 2–17 The titration curve of acetic acid. After addition of each
titrated with a solution of a strong base, usually sodium increment of NaOH to the acetic acid solution, the pH of the mixture is
hydroxide (NaOH), of known concentration. The NaOH measured. This value is plotted against the amount of NaOH added,
is added in small increments until the acid is consumed expressed as a fraction of the total NaOH required to convert all the
(neutralized), as determined with an indicator dye or a acetic acid (CH3COOH) to its deprotonated form, acetate (CH3COO2).
pH meter. The concentration of the acid in the original The points so obtained yield the titration curve. Shown in the boxes are
solution can be calculated from the volume and concen- the predominant ionic forms at the points designated. At the midpoint of
tration of NaOH added. The amounts of acid and base in the titration, the concentrations of the proton donor and proton acceptor
titrations are often expressed in terms of equivalents, are equal, and the pH is numerically equal to the pKa. The shaded zone
where one equivalent is the amount of a substance that is the useful region of buffering power, generally between 10% and 90%
will react with, or supply, one mole of hydrogen ions in titration of the weak acid.
an acid-base reaction.
A plot of pH against the amount of NaOH added (a further to satisfy its own equilibrium constant (Eqn 2–8).
titration curve), reveals the pKa of the weak acid. The net result as the titration proceeds is that more and
Consider the titration of a 0.1 M solution of acetic acid more HAc ionizes, forming Ac2, as the NaOH is added.
with 0.1 M NaOH at 25 8C (Fig. 2–17). Two reversible At the midpoint of the titration, at which exactly 0.5
equilibria are involved in the process (here, for simplic- equivalent of NaOH has been added per equivalent of
ity, acetic acid is denoted HAc): the acid, one-half of the original acetic acid has under-
gone dissociation, so that the concentration of the proton
H2O ∆ H1 1 OH2 (2–5)
donor, [HAc], now equals that of the proton acceptor,
HAc ∆ H1 1 Ac2 (2–6) [Ac2]. At this midpoint a very important relationship
holds: the pH of the equimolar solution of acetic acid
The equilibria must simultaneously conform to their
and acetate is exactly equal to the pKa of acetic acid
characteristic equilibrium constants, which are, respec-
(pKa 5 4.76; Figs 2–16, 2–17). The basis for this rela-
tively,
tionship, which holds for all weak acids, will soon
Kw 5 [H1][OH2 4 5 1 3 10214 M2 (2–7) become clear.
As the titration is continued by adding further
[H ][Ac ]
1 2
Ka 5 5 1.74 3 1025 M (2–8) increments of NaOH, the remaining nondissociated ace-
[HAc]
tic acid is gradually converted into acetate. The end
At the beginning of the titration, before any NaOH is point of the titration occurs at about pH 7.0: all the ace-
added, the acetic acid is already slightly ionized, to an tic acid has lost its protons to OH2, to form H2O and
extent that can be calculated from its ionization con- acetate. Throughout the titration the two equilibria
stant (Eqn 2–8). (Eqns 2–5, 2–6) coexist, each always conforming to its
As NaOH is gradually introduced, the added OH2 equilibrium constant.
combines with the free H1 in the solution to form H2O, Figure 2–18 compares the titration curves of
to an extent that satisfies the equilibrium relationship in three weak acids with very different ionization con-
Equation 2–7. As free H1 is removed, HAc dissociates stants: acetic acid (pKa 5 4.76); dihydrogen phosphate,
64 Water
Kw ⫽ [H⫹][OH⫺]
Buffers Are Mixtures of Weak Acids and Their
Conjugate Bases OH⫺ H2O
Buffers are aqueous systems that tend to resist changes
in pH when small amounts of acid (H1) or base (OH2)
are added. A buffer system consists of a weak acid (the
Acetic acid Acetate
proton donor) and its conjugate base (the proton accep- HAc Ac⫺
(CH3COOH) (CH3COO⫺)
tor). As an example, a mixture of equal concentrations
of acetic acid and acetate ion, found at the midpoint of
the titration curve in Figure 2–17, is a buffer system.
Notice that the titration curve of acetic acid has a rela- H⫹
tively flat zone extending about 1 pH unit on either side [H⫹][Ac⫺]
of its midpoint pH of 4.76. In this zone, a given amount Ka ⫽
[HAc]
of H1 or OH2 added to the system has much less effect
on pH than the same amount added outside the zone. FIGURE 2–19 The acetic acid–acetate pair as a buffer system. The sys-
tem is capable of absorbing either H1 or OH2 through the reversibility of
This relatively flat zone is the buffering region of the
the dissociation of acetic acid. The proton donor, acetic acid (HAc), con-
acetic acid–acetate buffer pair. At the midpoint of the
tains a reserve of bound H1, which can be released to neutralize an
buffering region, where the concentration of the proton
addition of OH2 to the system, forming H2O. This happens because the
donor (acetic acid) exactly equals that of the proton
product [H1][OH2] transiently exceeds Kw (1 3 10214 M2). The equilib-
acceptor (acetate), the buffering power of the system is
rium quickly adjusts to restore the product to 1 3 10214 M2 (at 25 8C),
maximal; that is, its pH changes least on addition of H1 thus transiently reducing the concentration of H1. But now the quotient
or OH2. The pH at this point in the titration curve of [H1][Ac2]/[HAc] is less than Ka, so HAc dissociates further to restore
acetic acid is equal to its pKa. The pH of the acetate buf- equilibrium. Similarly, the conjugate base, Ac2, can react with H1 ions
fer system does change slightly when a small amount of added to the system; again, the two ionization reactions simultaneously
H1 or OH2 is added, but this change is very small com- come to equilibrium. Thus a conjugate acid-base pair, such as acetic
pared with the pH change that would result if the same acid and acetate ion, tends to resist a change in pH when small amounts of
amount of H1 or OH2 were added to pure water or to a acid or base are added. Buffering action is simply the consequence of two
solution of the salt of a strong acid and strong base, reversible reactions taking place simultaneously and reaching their points
such as NaCl, which has no buffering power. of equilibrium as governed by their equilibrium constants, Kw and Ka.
Buffering results from two reversible reaction equi-
libria occurring in a solution of nearly equal concentra-
tions of a proton donor and its conjugate proton accep- sues of vertebrates. This equation is simply a useful way
tor. Figure 2–19 explains how a buffer system works. of restating the expression for the ionization constant of
Whenever H1 or OH2 is added to a buffer, the result is an acid. For the ionization of a weak acid HA, the Hen-
a small change in the ratio of the relative concentrations derson-Hasselbalch equation can be derived as follows:
of the weak acid and its anion and thus a small change
in pH. The decrease in concentration of one component [H1][A2]
Ka 5
of the system is balanced exactly by an increase in the [HA]
other. The sum of the buffer components does not First solve for [H1]:
change, only their ratio.
Each conjugate acid-base pair has a characteristic [HA]
[H1] 5 Ka
pH zone in which it is an effective buffer (Fig. 2–18). [A2]
22
The H2PO2 4 /HPO4 pair has a pKa of 6.86 and thus can Then take the negative logarithm of both sides:
serve as an effective buffer system between approxi-
mately pH 5.9 and pH 7.9; the NH41/NH3 pair, with a pKa [HA]
2log [H1] 5 2log Ka 2 log
of 9.25, can act as a buffer between approximately pH 8.3 [A2]
and pH 10.3. Substitute pH for 2log [H1] and pKa for 2log Ka:
[HA]
pH 5 pKa 2 log
The Henderson-Hasselbalch Equation Relates pH, pKa, [A2]
and Buffer Concentration Now invert 2log [HA]/[A2], which involves changing its
The titration curves of acetic acid, H2PO2
4, and NH41 sign, to obtain the Henderson-Hasselbalch equation:
(Fig. 2–18) have nearly identical shapes, suggesting that
[A2]
these curves reflect a fundamental law or relationship. pH 5 pKa 1 log (2–9)
[HA]
This is indeed the case. The shape of the titration curve
of any weak acid is described by the Henderson-Hassel- This equation fits the titration curve of all weak acids
balch equation, which is important for understanding and enables us to deduce some important quantitative
buffer action and acid-base balance in the blood and tis- relationships. For example, it shows why the pKa of a
2.3 Buffering against pH Changes in Biological Systems 65
dissociated is the imidazole group, which can be proton-

Protein Protein
A A ated (we’ll abbreviate as HisH1) or not (His).
CH2 CH2 We use the Henderson-Hasselbalch equation:
A NH 3::4 A NH
C C [A2]
CH CH ⫹ H⫹ pH 5 pKa 1 log
[HA]
HC ⫹ HC
N N Substituting pK2 5 6.0 and pH 5 7.3:
H
pH 5 pH 7 [His]
7.3 5 6.0 1 log
[HisH1]
FIGURE 2–20 Ionization of histidine. The amino acid histidine, a compo-
[His]
nent of proteins, is a weak acid. The pKa of the protonated nitrogen of 1.3 5 log
the side chain is 6.0. [HisH1]
[His]
antilog 1.3 5 5 2.0 3 101
[HisH1]
weak acid is equal to the pH of the solution at the mid-
point of its titration. At that point, [HA] 5 [A2], and This gives us the ratio of [His] to [HisH1] (20 to 1 in this
case). We want to convert this ratio to the fraction of
pH 5 pKa 1 log 1 5 pKa 1 0 5 pKa total histidine that is in the unprotonated form His at
The Henderson-Hasselbalch equation also allows us to pH 7.3. That fraction is 20/21 (20 parts His per 1 part
(1) calculate pKa, given pH and the molar ratio of pro- HisH1, in a total of 21 parts histidine in either form),
ton donor and acceptor; (2) calculate pH, given pKa and or about 95.2%; the remainder (100% minus 95.2%) is
the molar ratio of proton donor and acceptor; and protonated—about 5%.
(3) calculate the molar ratio of proton donor and accep-
tor, given pH and pKa. Nucleotides such as ATP, as well as many metabo-
lites of low molecular weight, contain ionizable groups
Weak Acids or Bases Buffer Cells and Tissues against that can contribute buffering power to the cytoplasm.
pH Changes Some highly specialized organelles and extracellular
compartments have high concentrations of compounds
The intracellular and extracellular fluids of multicellular that contribute buffering capacity: organic acids buffer
organisms have a characteristic and nearly constant pH. the vacuoles of plant cells; ammonia buffers urine.
The organism’s first line of defense against changes in Two especially important biological buffers are the
internal pH is provided by buffer systems. The cytoplasm phosphate and bicarbonate systems. The phosphate buffer
of most cells contains high concentrations of proteins, and system, which acts in the cytoplasm of all cells, consists of
these proteins contain many amino acids with functional H2PO2 22
4 as proton donor and HPO4 as proton acceptor:
groups that are weak acids or weak bases. For example,
22
the side chain of histidine (Fig. 2–20) has a pKa of 6.0 H2PO2
4 ∆ H 1 HPO4
1
and thus can exist in either the protonated or unproton- The phosphate buffer system is maximally effective at a
ated form near neutral pH. Proteins containing histidine pH close to its pKa of 6.86 (Figs 2–16, 2–18) and thus tends
residues therefore buffer effectively near neutral pH. to resist pH changes in the range between about 5.9 and
7.9. It is therefore an effective buffer in biological fluids; in
mammals, for example, extracellular fluids and most cyto-
WORKED EXAMPLE 2–5 Ionization of Histidine plasmic compartments have a pH in the range of 6.9 to 7.4.
Calculate the fraction of histidine that has its imidazole
side chain protonated at pH 7.3. The pKa values for WORKED EXAMPLE 2–6 Phosphate Buffers
histidine are pK1 5 1.8, pK2 (imidazole) 5 6.0, and
(a) What is the pH of a mixture of 0.042 M NaH2PO4 and
pK3 5 9.2 (see Fig. 3–12b).
0.058 M Na2HPO4?
Solution: The three ionizable groups in histidine have suffi-
Solution: We use the Henderson-Hasselbalch equation,
ciently different pKa values that the first acid (—COOH) is
which we’ll express here as
completely ionized before the second (protonated imidaz-
ole) begins to dissociate a proton, and the second ionizes [conjugate base]
pH 5 pKa 1 log
completely before the third (—NH13 ) begins to dissociate [acid]
its proton. (With the Henderson-Hasselbalch equation, we
In this case, the acid (the species that gives up a proton)
can easily show that a weak acid goes from 1% ionized at
is H2PO2 4 , and the conjugate base (the species that gains
2 pH units below its pKa to 99% ionized at 2 pH units above
a proton) is HPO22 4 . Substituting the given concentra-
its pKa; see also Fig. 3–12b.) At pH 7.3, the carboxyl group
tions of acid and conjugate base and the pKa (6.86),
of histidine is entirely deprotonated (—COO2) and the
a-amino group is fully protonated (—NH13 ). We can there- 0.058
pH 5 6.86 1 log 5 6.86 1 0.14 5 7.0
fore assume that at pH 7.3, the only group that is partially 0.042
66 Water
We can roughly check this answer. When more con- in turn depends on the concentration of dissolved CO2,
jugate base than acid is present, the acid is more than which in turn depends on the concentration of CO2 in
50% titrated and thus the pH is above the pKa (6.86), the gas phase, or the partial pressure of CO2, denoted
where the acid is exactly 50% titrated. pCO2. Thus the pH of a bicarbonate buffer exposed to a
gas phase is ultimately determined by the concentration
(b) If 1.0 mL of 10.0 M NaOH is added to a liter of the
of HCO2 3 in the aqueous phase and by pCO2 in the gas
buffer prepared in (a), how much will the pH change?
phase.
Solution: A liter of the buffer contains 0.042 mol of The bicarbonate buffer system is an effective
NaH2PO4. Adding 1.0 mL of 10.0 M NaOH (0.010 mol) physiological buffer near pH 7.4, because the
would titrate an equivalent amount (0.010 mol) of H2CO3 of blood plasma is in equilibrium with a large
NaH2PO4 to Na2HPO4, resulting in 0.032 mol of NaH2PO4 reserve capacity of CO2(g) in the air space of the lungs.
and 0.068 mol of Na2HPO4. The new pH is As noted above, this buffer system involves three
reversible equilibria, in this case between gaseous CO2
[HPO224 ] in the lungs and bicarbonate (HCO2
pH 5 pKa 1 log 3 ) in the blood
[H2PO2 4] plasma (Fig. 2–21).
0.068 Blood can pick up H1, such as from the lactic acid
5 6.86 1 log 5 6.86 1 0.33 5 7.2
0.032 produced in muscle tissue during vigorous exercise.
(c) If 1.0 mL of 10.0 M NaOH is added to a liter of pure Alternatively, it can lose H1, such as by protonation of
water at pH 7.0, what is the final pH? Compare this with the NH3 produced during protein catabolism. When H1
the answer in (b). is added to blood as it passes through the tissues, reac-
tion 1 in Figure 2–21 proceeds toward a new equilibrium,
Solution: The NaOH dissociates completely into Na1 and in which [H2CO3] is increased. This in turn increases
OH2, giving [OH2] 5 0.010 mol/L 5 1.0 3 1022 M. The [CO2(d)] in the blood (reaction 2) and thus increases
pOH is the negative logarithm of [OH2], so pOH 5 2.0. the partial pressure of CO2(g) in the air space of the
Given that in all solutions, pH 1 pOH 5 14, the pH of lungs (reaction 3); the extra CO2 is exhaled. Conversely,
the solution is 12. when H1 is lost from the blood, the opposite events
So, an amount of NaOH that increases the pH of occur: more H2CO3 dissociates into H1 and HCO2 3 and
water from 7 to 12 increases the pH of a buffered solu- thus more CO2(g) from the lungs dissolves in blood
tion, as in (b), from 7.0 to just 7.2. Such is the power of plasma. The rate of respiration—that is, the rate of inhal-
buffering! ing and exhaling—can quickly adjust these equilibria
to keep the blood pH nearly constant. The rate of respi-
Blood plasma is buffered in part by the bicarbonate ration is controlled by the brain stem, where detection
system, consisting of carbonic acid (H2CO3) as proton of an increased blood pCO2 or decreased blood pH trig-
donor and bicarbonate (HCO2 3 ) as proton acceptor gers deeper and more frequent breathing.
(K1 is the first of several equilibrium constants in the At the pH of blood plasma (7.4) very little H2CO3 is
bicarbonate buffering system): present in comparison with HCO2 3 , and the addition of a
H2CO3 ∆ H1 1 HCO2 small amount of base (NH3 or OH2) would titrate this
3
[H1][HCO2 H2CO3, exhausting the buffering capacity. The important
3]
K1 5
[H2CO3]
This buffer system is more complex than other conjugate
acid-base pairs because one of its components, carbonic H HCO 3
acid (H2CO3), is formed from dissolved (d) carbon diox- reaction 1
ide and water, in a reversible reaction:
H2CO3
Aqueous phase
CO2 (d) 1 H2O ∆ H2CO3
(blood in capillaries) reaction 2
[H2CO3]
K2 5 H 2O H2O
[CO2 (d)][H2O]
CO2(d)
Carbon dioxide is a gas under normal conditions, and
reaction 3
CO2 dissolved in an aqueous solution is in equilibrium
with CO2 in the gas (g) phase: Gas phase
(lung air space) CO2(g)
CO2 (g) ∆ CO2 (d)

[CO2 (d)] FIGURE 2–21 The bicarbonate buffer system. CO2 in the air space of
K3 5 the lungs is in equilibrium with the bicarbonate buffer in the blood
[CO2 (g)]
plasma passing through the lung capillaries. Because the concentration
The pH of a bicarbonate buffer system depends on the of dissolved CO2 can be adjusted rapidly through changes in the rate of
concentration of H2CO3 and HCO2 3 , the proton donor breathing, the bicarbonate buffer system of the blood is in near-equilibrium
and acceptor components. The concentration of H2CO3 with a large potential reservoir of CO2.
7
Carbohydrates and Glycobiology
7.1 Monosaccharides and Disaccharides 243 D-glucose, sometimes referred to as dextrose. Monosac-
charides of four or more carbons tend to have cyclic
7.2 Polysaccharides 254 structures.
7.3 Glycoconjugates: Proteoglycans, Glycoproteins, Oligosaccharides consist of short chains of mono-
and Glycosphingolipids 263 saccharide units, or residues, joined by characteristic
linkages called glycosidic bonds. The most abundant are
7.4 Carbohydrates as Informational Molecules: the disaccharides, with two monosaccharide units.
The Sugar Code 269 Typical is sucrose (cane sugar), which consists of the
7.5 Working with Carbohydrates 274 six-carbon sugars D-glucose and D-fructose. All common
monosaccharides and disaccharides have names ending
with the suffix “-ose.” In cells, most oligosaccharides
C
arbohydrates are the most abundant biomolecules
on Earth. Each year, photosynthesis converts more consisting of three or more units do not occur as free
than 100 billion metric tons of CO2 and H2O into entities but are joined to nonsugar molecules (lipids or
cellulose and other plant products. Certain carbohy- proteins) in glycoconjugates.
drates (sugar and starch) are a dietary staple in most The polysaccharides are sugar polymers contain-
parts of the world, and the oxidation of carbohydrates ing more than 20 or so monosaccharide units; some
is the central energy-yielding pathway in most nonpho- have hundreds or thousands of units. Some polysac-
tosynthetic cells. Carbohydrate polymers (also called charides, such as cellulose, are linear chains; others,
glycans) serve as structural and protective elements in such as glycogen, are branched. Both glycogen and
the cell walls of bacteria and plants and in the connec- cellulose consist of recurring units of D-glucose, but
tive tissues of animals. Other carbohydrate polymers they differ in the type of glycosidic linkage and conse-
lubricate skeletal joints and participate in recognition quently have strikingly different properties and bio-
and adhesion between cells. Complex carbohydrate logical roles.
polymers covalently attached to proteins or lipids act
as signals that determine the intracellular destination
or metabolic fate of these hybrid molecules, called gly-
7.1 Monosaccharides and Disaccharides
coconjugates. This chapter introduces the major The simplest of the carbohydrates, the monosaccha-
classes of carbohydrates and glycoconjugates and pro- rides, are either aldehydes or ketones with two or
vides a few examples of their many structural and func- more hydroxyl groups; the six-carbon monosaccha-
tional roles. rides glucose and fructose have five hydroxyl groups.
Carbohydrates are polyhydroxy aldehydes or Many of the carbon atoms to which hydroxyl groups
ketones, or substances that yield such compounds on are attached are chiral centers, which give rise to the
hydrolysis. Many, but not all, carbohydrates have the many sugar stereoisomers found in nature. Stereo-
empirical formula (CH2O)n; some also contain nitrogen, isomerism in sugars is biologically significant because
phosphorus, or sulfur. There are three major size classes the enzymes that act on sugars are strictly stereo-
of carbohydrates: monosaccharides, oligosaccharides, specific, typically preferring one stereoisomer to
and polysaccharides (the word “saccharide” is derived another by three or more orders of magnitude, as
from the Greek sakcharon, meaning “sugar”). Mono- reflected in Km values or binding constants. It is as
saccharides, or simple sugars, consist of a single difficult to fit the wrong sugar stereoisomer into an
polyhydroxy aldehyde or ketone unit. The most abun- enzyme’s binding site as it is to put your left glove on
dant monosaccharide in nature is the six-carbon sugar your right hand.
243
244 Carbohydrates and Glycobiology
We begin by describing the families of monosac- Monosaccharides with four, five, six, and seven car-
charides with backbones of three to seven carbons— bon atoms in their backbones are called, respectively,
their structure and stereoisomeric forms, and the tetroses, pentoses, hexoses, and heptoses. There are
means of representing their three-dimensional struc- aldoses and ketoses of each of these chain lengths: aldo-
tures on paper. We then discuss several chemical reac- tetroses and ketotetroses, aldopentoses and ketopen-
tions of the carbonyl groups of monosaccharides. One toses, and so on. The hexoses, which include the aldo-
such reaction, the addition of a hydroxyl group from hexose D-glucose and the ketohexose D-fructose (Fig.
within the same molecule, generates cyclic forms hav- 7–1b), are the most common monosaccharides in
ing four or more backbone carbons (the forms that nature—the products of photosynthesis, and key inter-
predominate in aqueous solution). This ring closure mediates in the central energy-yielding reaction
creates a new chiral center, adding further stereo- sequence in most organisms. The aldopentoses D-ribose
chemical complexity to this class of compounds. The and 2-deoxy-D-ribose (Fig. 7–1c) are components of
nomenclature for unambiguously specifying the con- nucleotides and nucleic acids (Chapter 8).
figuration about each carbon atom in a cyclic form and
the means of representing these structures on paper
are therefore described in some detail; this informa-
Monosaccharides Have Asymmetric Centers
tion will be useful as we discuss the metabolism of All the monosaccharides except dihydroxyacetone
monosaccharides in Part II. We also introduce here contain one or more asymmetric (chiral) carbon atoms
some important monosaccharide derivatives encoun- and thus occur in optically active isomeric forms
tered in later chapters. (pp. 17–18). The simplest aldose, glyceraldehyde, con-
tains one chiral center (the middle carbon atom) and
therefore has two different optical isomers, or enan-
The Two Families of Monosaccharides Are Aldoses tiomers (Fig. 7–2).
and Ketoses
Monosaccharides are colorless, crystalline solids that KEY CONVENTION: One of the two enantiomers of glycer-
are freely soluble in water but insoluble in nonpolar aldehyde is, by convention, designated the D isomer,
solvents. Most have a sweet taste (see Box 7–2, p. 254). the other the L isomer. As for other biomolecules with
The backbones of common monosaccharides are chiral centers, the absolute configurations of sugars
unbranched carbon chains in which all the carbon are known from x-ray crystallography. To represent
atoms are linked by single bonds. In this open-chain three-dimensional sugar structures on paper, we often
form, one of the carbon atoms is double-bonded to an use Fischer projection formulas (Fig. 7–2). In
oxygen atom to form a carbonyl group; each of the other Fischer projection formulas, horizontal bonds project
carbon atoms has a hydroxyl group. If the carbonyl out of the plane of the paper, toward the reader; verti-
group is at an end of the carbon chain (that is, in an cal bonds project behind the plane of the paper, away
aldehyde group) the monosaccharide is an aldose; if from the reader. ■
the carbonyl group is at any other position (in a ketone
group) the monosaccharide is a ketose. The simplest In general, a molecule with n chiral centers can
monosaccharides are the two three-carbon trioses: glyc- have 2n stereoisomers. Glyceraldehyde has 21 5 2; the
eraldehyde, an aldotriose, and dihydroxyacetone, a aldohexoses, with four chiral centers, have 24 5 16. The
ketotriose (Fig. 7–1a). stereoisomers of monosaccharides of each carbon-chain
H O H
C H C OH H O H O
H O H H C OH C O C C
C H C OH HO C H HO C H H C OH CH 2
H C OH C O H C OH H C OH H C OH H C OH
A A
H C OH H C OH H C OH H C OH H C OH H C OH
H H CH2OH CH 2OH CH 2OH CH2OH
D-Glyceraldehyde, Dihydroxyacetone, D-Glucose, D-Fructose, D-Ribose, 2-Deoxy-D-ribose,
an aldotriose a ketotriose an aldohexose a ketohexose an aldopentose an aldopentose
(a) (b) (c)
FIGURE 7–1 Representative monosaccharides. (a) Two trioses, an component of ribonucleic acid (RNA), and 2-deoxy-D-ribose is a compo-
aldose and a ketose. The carbonyl group in each is shaded. (b) Two com- nent of deoxyribonucleic acid (DNA).
mon hexoses. (c) The pentose components of nucleic acids. D-Ribose is a
7.1 Monosaccharides and Disaccharides 245
Figure 7–3 shows the structures of the D stereo-

Mirror isomers of all the aldoses and ketoses having three to
CHO CHO six carbon atoms. The carbons of a sugar are numbered
beginning at the end of the chain nearest the carbonyl
group. Each of the eight D-aldohexoses, which differ in
H
OH the stereochemistry at C-2, C-3, or C-4, has its own
H OH
name: D-glucose, D-galactose, D-mannose, and so forth
(Fig. 7–3a). The four- and five-carbon ketoses are
designated by inserting “ul” into the name of a corre-
CH2OH sponding aldose; for example, D-ribulose is the keto-
CH2OH
pentose corresponding to the aldopentose D-ribose.
(We will see the importance of ribulose when we dis-
cuss the fixation of atmospheric CO2 by green plants,
Ball-and-stick models in Chapter 20.) The ketohexoses are named otherwise:
for example, fructose (from the Latin fructus, “fruit”;
CHO CHO fruits are one source of this sugar) and sorbose (from
H C OH HO C H Sorbus, the genus of mountain ash, which has berries
rich in the related sugar alcohol sorbitol). Two sugars
CH2OH CH2OH
that differ only in the configuration around one carbon
D-Glyceraldehyde L-Glyceraldehyde
atom are called epimers; D-glucose and D-mannose,
Fischer projection formulas which differ only in the stereochemistry at C-2, are
epimers, as are D-glucose and D-galactose (which differ
CHO CHO at C-4) (Fig. 7–4).
Some sugars occur naturally in their L form; exam-
H C OH HO C H
ples are L-arabinose and the L isomers of some sugar
CH 2OH CH2OH derivatives that are common components of glycoconju-
D-Glyceraldehyde L-Glyceraldehyde gates (Section 7.3).
Perspective formulas
FIGURE 7–2 Three ways to represent the two enantiomers of glyceral- H O
dehyde. The enantiomers are mirror images of each other. Ball-and-stick G J
C
models show the actual configuration of molecules. Recall (see Fig. 1–18) A
HOCOOH
that in perspective formulas, the wide end of a solid wedge projects out A
of the plane of the paper, toward the reader; a dashed wedge extends HOOCOH
A
behind.
HOOCOH
A
CH2OH
L-Arabinose
length can be divided into two groups that differ in the
configuration about the chiral center most distant
from the carbonyl carbon. Those in which the configu-
ration at this reference carbon is the same as that of
The Common Monosaccharides Have
D-glyceraldehyde are designated D isomers, and those Cyclic Structures
with the same configuration as L-glyceraldehyde are L For simplicity, we have thus far represented the struc-
isomers. In other words, when the hydroxyl group on tures of aldoses and ketoses as straight-chain molecules
the reference carbon is on the right (dextro) in a pro- (Figs 7–3, 7–4). In fact, in aqueous solution, aldotetro-
jection formula that has the carbonyl carbon at the top, ses and all monosaccharides with five or more carbon
the sugar is the D isomer; when on the left (levo), it is atoms in the backbone occur predominantly as cyclic
the L isomer. Of the 16 possible aldohexoses, eight are (ring) structures in which the carbonyl group has
D forms and eight are L. Most of the hexoses of living formed a covalent bond with the oxygen of a hydroxyl
organisms are D isomers. Why D isomers? An interesting group along the chain. The formation of these ring
and unanswered question. Recall that all of the amino structures is the result of a general reaction between
acids found in protein are exclusively one of two pos- alcohols and aldehydes or ketones to form derivatives
sible stereoisomers, L. The basis for this initial prefer- called hemiacetals or hemiketals. Two molecules of
ence for one isomer during evolution is also unknown; an alcohol can add to a carbonyl carbon; the product of
however, once one isomer had been selected, it was the first addition is a hemiacetal (for addition to an
likely that evolving enzymes would retain their prefer- aldose) or a hemiketal (for addition to a ketose). If the
ence for that stereoisomer (p. 78). —OH and carbonyl groups are from the same molecule,
(a) D-Aldoses
Three carbons Four carbons Five carbons

H O H O H O H O
H O H O C C C C
H O C C H C OH HO C H H C OH HO C H
C H C OH HO C H H C OH H C OH HO C H HO C H
H C OH H C OH H C OH H C OH H C OH H C OH H C OH
CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH
D-Glyceraldehyde D-Erythrose D-Threose D-Ribose D-Arabinose D-Xylose D-Lyxose
Six carbons
H O H O H O H O H O H O H O H O
C C C C C C C C
H C OH HO C H H C OH HO C H H C OH HO C H H C OH HO C H
H C OH H C OH HO C H HO C H H C OH H C OH HO C H HO C H
H C OH H C OH H C OH H C OH HO C H HO C H HO C H HO C H
H C OH H C OH H C OH H C OH H C OH H C OH H C OH H C OH
CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH
D-Allose D-Altrose D-Glucose D-Mannose D-Gulose D-Idose D-Galactose D-Talose
FIGURE 7–3 Aldoses and ketoses. The series of (a) D-aldoses and
(b) D-ketoses having from three to six carbon atoms, shown as projec-
(b) D-Ketoses tion formulas. The carbon atoms in red are chiral centers. In all these D
Three carbons Four carbons isomers, the chiral carbon most distant from the carbonyl carbon has the
same configuration as the chiral carbon in D-glyceraldehyde. The sugars
CH2OH named in boxes are the most common in nature; you will encounter
CH2OH C O these again in this and later chapters.
C O H C OH
CH2OH CH2OH
Dihydroxyacetone D-Erythrulose a five- or six-membered ring results. Addition of the
second molecule of alcohol produces the full acetal or
ketal (Fig. 7–5), and the bond formed is a glycosidic
Five carbons Six carbons linkage. When the two molecules that react are both
monosaccharides, the acetal or ketal formed is a disac-
CH2OH CH2OH
charide.
CH2OH C O C O The reaction with the first molecule of alcohol cre-
C O H C OH HO C H ates an additional chiral center (the carbonyl carbon).
H C OH H C OH H C OH Because the alcohol can add in either of two ways,
attacking either the “front” or the “back” of the car-
H C OH H C OH H C OH
bonyl carbon, the reaction can produce either of two
CH2OH CH2OH CH2OH stereoisomeric configurations, denoted ␣ and ␤. For
D-Ribulose D-Psicose D-Fructose example, D-glucose exists in solution as an intramolecu-
lar hemiacetal in which the free hydroxyl group at C-5
CH2OH CH2OH has reacted with the aldehydic C-1, rendering the latter
CH2OH C O C O carbon asymmetric and producing two possible stereo-
C O H C OH HO C H isomers, designated ␣ and ␤ (Fig. 7–6). Isomeric forms
of monosaccharides that differ only in their configura-
HO C H HO C H HO C H
tion about the hemiacetal or hemiketal carbon atom are
H C OH H C OH H C OH called anomers, and the carbonyl carbon atom is called
CH2OH CH2OH CH2OH the anomeric carbon.
D-Xylulose D-Sorbose D-Tagatose Six-membered ring compounds are called pyranoses
because they resemble the six-membered ring compound
1 1 1
CHO CHO CHO H O
2 2 2
HO C H H C OH H C OH 1C
3 3 3 2
HO C H HO C H HO C H H C OH
4 4 4 3
H C OH H C OH HO C H HO C H D-Glucose
5 5 5 4
H C OH H C OH H C OH H C OH
6 6 6 5
CH2OH CH2OH CH2OH H C OH
D-Mannose D-Glucose D-Galactose 6
CH2OH
(epimer at C-2) (epimer at C-4)
FIGURE 7–4 Epimers. D-Glucose and two of its epimers are shown as
6
projection formulas. Each epimer differs from D-glucose in the configura- CH2OH
tion at one chiral center (shaded light red or blue). 5C OH
H
:
H
4 H
C C1
OH H
HO O
3
C 2
C
3 3 H OH
O OH HO R OR
1 2 1 2 1 2
R C ⫹ HO R R C OR R C OR ⫹ HOH
H H 3
H
HO R 6
CH2OH 6
CH2OH
Aldehyde Alcohol Hemiacetal Acetal
5 5
C O C O
H H H OH
4 H 4 H
4 4 C 1C C 1C
OH HO R OR OH H OH H
HO OH HO H
1 3 1 3 1 3
R C O ⫹ HO R R C OR R C OR ⫹ HOH 3
C 2
C 3
C 2
C
2 2 4 2
R R HO R R H OH H OH
Ketone Alcohol Hemiketal Ketal ␣-D-Glucopyranose ␤-D-Glucopyranose
FIGURE 7–5 Formation of hemiacetals and hemiketals. An aldehyde FIGURE 7–6 Formation of the two cyclic forms of D-glucose. Reaction
or ketone can react with an alcohol in a 1:1 ratio to yield a hemiacetal or between the aldehyde group at C-1 and the hydroxyl group at C-5 forms
hemiketal, respectively, creating a new chiral center at the carbonyl carbon. a hemiacetal linkage, producing either of two stereoisomers, the ␣ and ␤
Substitution of a second alcohol molecule produces an acetal or ketal. anomers, which differ only in the stereochemistry around the hemiacetal
When the second alcohol is part of another sugar molecule, the bond carbon. This reaction is reversible. The interconversion of ␣ and ␤ ano-
produced is a glycosidic bond (p. 252). mers is called mutarotation.
pyran (Fig. 7–7). The systematic names for the two ring place the hydroxyl groups. If a hydroxyl group is to
forms of D-glucose are therefore ␣-D-glucopyranose and the right in the Fischer projection, it is placed pointing
␤-D-glucopyranose. Ketohexoses (such as fructose) also down (i.e., below the plane of the ring) in the Haworth
occur as cyclic compounds with ␣ and ␤ anomeric forms. perspective; if it is to the left in the Fischer projection,
In these compounds the hydroxyl group at C-5 (or C-6) it is placed pointing up (i.e., above the plane) in the
reacts with the keto group at C-2, forming a furanose (or Haworth perspective. The terminal —CH2OH group
pyranose) ring containing a hemiketal linkage (Fig. 7–5). projects upward for the D-enantiomer, downward for
D-Fructose readily forms the furanose ring (Fig. 7–7); the the L-enantiomer. The hydroxyl on the anomeric carbon
more common anomer of this sugar in combined forms or can point up or down. When the anomeric hydroxyl
in derivatives is ␤-D-fructofuranose. of a D-hexose is on the same side of the ring as C-6, the
Cyclic sugar structures are more accurately repre- structure is by definition ␤; when it is on the opposite
sented in Haworth perspective formulas than in the side from C-6, the structure is ␣. ■
Fischer projections commonly used for linear sugar
structures. In Haworth projections the six-membered
ring is tilted to make its plane almost perpendicular to
1
that of the paper, with the bonds closest to the reader CHO
drawn thicker than those farther away, as in Figure 7–7. H
2
C OH
6 CH
2OH
3 5 O
HO C H H H
KEY CONVENTION: To convert the Fischer projection for- 4
H
H C OH 4 1
mula of any linear D-hexose to a Haworth perspective OH H
5 HO OH
formula showing the molecule’s cyclic structure, draw H C OH 3 2
the six-membered ring (five carbons and one oxygen, 6
CH2OH H OH
at the upper right), number the carbons in a clockwise D-Glucose ␣-D-Glucopyranose
direction beginning with the anomeric carbon, then Fischer projection Haworth perspective
6 CH
2OH
5 O 6
WORKED EXAMPLE 7–2 Drawing Haworth Perspective
H H H HOCH2 O 1 CH
2OH Formulas of Sugar Isomers
4 1 5 2
OH H
OH
H HO Draw the Haworth perspective formulas for ␣-D-mannose
HO H OH
3 2 4 3 and ␤-L-galactose.
H OH OH H
␣-D-Glucopyranose ␣-D-Fructofuranose Solution: The Haworth perspective formula of D-mannose
from Worked Example 7–1 can have the hydroxyl group
at C-1 pointing either up or down. According to the Key
CH2OH Convention, for the ␣ form, the C-1 hydroxyl is pointing
O
H OH HOCH2 O OH down when C-6 is up, as it is in D-mannose.
H
For ␤-L-galactose, use the Fischer representation
OH H H HO
HO H H CH2OH of D-galactose (see Worked Example 7–1) to draw the
correct Fischer representation of L-galactose, which
H OH OH H is its mirror image: the hydroxyls at C-2, C-3, C-4, and
␤-D-Glucopyranose ␤-D-Fructofuranose C-5 are on the left, right, right, and left sides, respec-
tively. Now draw the Haworth perspective, a six-
HC O O membered ring in which the —OH groups on C-2, C-3,
HC
and C-4 are oriented up, down, and down, respec-
CH
HC CH tively, because in the Fischer representation they
C C are on the left, right, and right sides. Because it is the
H2C CH H H
␤ form, the —OH on the anomeric carbon points down
Pyran Furan (same side as C-5).
FIGURE 7–7 Pyranoses and furanoses. The pyranose forms of D-glucose
and the furanose forms of D-fructose are shown here as Haworth perspec-
tive formulas. The edges of the ring nearest the reader are represented by
bold lines. Hydroxyl groups below the plane of the ring in these Haworth
perspectives would appear at the right side of a Fischer projection (com-
pare with Fig. 7–6). Pyran and furan are shown for comparison. The ␣ and ␤ anomers of D-glucose interconvert in
aqueous solution by a process called mutarotation,
WORKED EXAMPLE 7–1 Conversion of Fischer Projection in which one ring form (say, the ␣ anomer) opens
briefly into the linear form, then closes again to pro-
to Haworth Perspective
duce the ␤ anomer (Fig. 7–6). Thus, a solution of
Formulas ␤-D-glucose and a solution of ␣-D-glucose eventually
Draw the Haworth perspective formulas for D-mannose form identical equilibrium mixtures having identical
and D-galactose. optical properties. This mixture consists of about
one-third ␣-D-glucose, two-thirds ␤-D-glucose, and
1
CHO
1
CHO very small amounts of the linear and five-membered
2 2 ring (glucofuranose) forms.
HO C H H C OH
3 3
Haworth perspective formulas like those in Figure
HO C H HO C H 7–7 are commonly used to show the stereochemistry
H
4
C OH HO
4
C H of ring forms of monosaccharides. However, the six-
H
5
C OH H
5
C OH
membered pyranose ring is not planar, as Haworth
6 6 perspectives suggest, but tends to assume either of
CH2OH CH2OH
two “chair” conformations (Fig. 7–8). Recall from
D-Mannose D-Galactose
Chapter 1 (pp. 18–19) that two conformations of a
molecule are interconvertible without the breakage of
Solution: Pyranoses are six-membered rings, so start with
covalent bonds, whereas two configurations can be
six-membered Haworth structures with the oxygen atom
interconverted only by breaking a covalent bond. To
at the top right. Number the carbon atoms clockwise,
interconvert ␣ and ␤ configurations, the bond involving
starting with the aldose carbon. For mannose, place the
the ring oxygen atom would have to be broken, but
hydroxyls on C-2, C-3, and C-4 above, above, and below
interconversion of the two chair forms (which are
the ring, respectively (because in the Fischer projection
conformers) does not require bond breakage and
they are on the left, left, and right sides of the mannose
does not change configurations at any of the ring car-
structure). For D-galactose, the hydroxyls are oriented
bons. The specific three-dimensional structures of the
below, above, and above for C-2, C-3, and C-4, respectively.
monosaccharide units are important in determining
The hydroxyl at C-1 can be either up or down; there are
the biological properties and functions of some poly-
two possible configurations, ␣ and ␤, at this carbon.
saccharides, as we shall see.
Axis Axis FIGURE 7–8 Conformational formulas of pyranoses. (a) Two chair
H CH2OH OH forms of the pyranose ring of ␤-D-glucopyranose. Two conformers such
CH2OH as these are not readily interconvertible; an input of about 46 kJ of ener-
O H
HO H OH O H
gy per mole of sugar is required to force the interconversion of chair
H OH H forms. Another conformation, the “boat” (not shown), is seen only in
HO H
OH H derivatives with very bulky substituents. (b) The preferred chair confor-
H H OH OH
mation of ␣-D-glucopyranose.
Two possible chair forms of ␤-D-glucopyranose
(a)
Organisms Contain a Variety of Hexose Derivatives
Axis
In addition to simple hexoses such as glucose, galactose,
H
CH2OH O and mannose, there are a number of sugar derivatives in
HO H which a hydroxyl group in the parent compound is
H replaced with another substituent, or a carbon atom is
HO H
OH oxidized to a carboxyl group (Fig. 7–9). In glucosamine,
H OH galactosamine, and mannosamine, the hydroxyl at C-2 of
␣-D-Glucopyranose the parent compound is replaced with an amino group.
(b) The amino group is commonly condensed with acetic
Glucose family
Amino sugars
CH2OH CH2OH CH2OH CH2OH CH2OH

O O O O O
H H OH H H OH H H OH HO H OH H H H N OH
2
OH H OH H OH H OH H OH
HO H HO H HO H H H HO H
H OH H NH2 H NH H NH2 H H
C O ␤-D-Galactosamine ␤-D-Mannosamine
CH3
␤-D-Glucose ␤-D-Glucosamine N-Acetyl-␤-D-glucosamine
Deoxy sugars
CH2 O PO2⫺
3 CH2OH CH2OH H H
O O O CH3 O O
H OH H OH H OH H CH3 H HO CH3 OH
H H H
R⫽ O C H
OH H R H R H H OH H H
HO H HO H HO H HO OH H H
COO⫺
H OH H NH2 H NH OH H OH OH
C O
CH3
␤-D-Glucose 6-phosphate Muramic acid N-Acetylmuramic acid ␤-L-Fucose ␣-L-Rhamnose
Acidic sugars
O O⫺ CH3 O O⫺
C CH2OH CH2OH O C H R⫽
C
O OH O O
H OH H O⫺ H HN
H H H R
C O H C OH
OH H OH H OH H H H
HO H HO O HO H OH H C OH
H OH H OH H OH OH H CH2OH
␤-D-Glucuronate D-Gluconate D-Glucono-␦-lactone N-Acetylneuraminic acid
(a sialic acid)
FIGURE 7–9 Some hexose derivatives important in biology. In amino sugars contain a carboxylate group, which confers a negative charge at
sugars, an —NH2 group replaces one of the —OH groups in the parent neutral pH. D-Glucono-␦-lactone results from formation of an ester linkage
hexose. Substitution of —H for —OH produces a deoxy sugar; note that between the C-1 carboxylate group and the C-5 (also known as the ␦
the deoxy sugars shown here occur in nature as the L isomers. The acidic carbon) hydroxyl group of D-gluconate.
acid, as in N-acetylglucosamine. This glucosamine corresponding uronic acid: glucuronic, galacturonic, or

derivative is part of many structural polymers, includ- mannuronic acid. Both aldonic and uronic acids form sta-
ing those of the bacterial cell wall. The substitution of ble intramolecular esters called lactones (Fig. 7–9, lower
a hydrogen for the hydroxyl group at C-6 of L-galactose left). The sialic acids are a family of sugars with the same
or L-mannose produces L-fucose or L-rhamnose, respec- nine-carbon backbone. One of them, N-acetylneuraminic
tively. L-Fucose is found in the complex oligosaccha- acid (often referred to simply as “sialic acid”), is a deriva-
ride components of glycoproteins and glycolipids; tive of N-acetylmannosamine that occurs in many glyco-
L-rhamnose is found in plant polysaccharides. proteins and glycolipids on animal cell surfaces, providing
Oxidation of the carbonyl (aldehyde) carbon of glu- sites of recognition by other cells or extracellular carbohy-
cose to the carboxyl level produces gluconic acid, used in drate-binding proteins. The carboxylic acid groups of the
medicine as an innocuous counterion with which to admin- acidic sugar derivatives are ionized at pH 7, and the com-
ister positively charged drugs (such as quinine) or ions pounds are therefore correctly named as the carboxyl-
(such as Ca21). Other aldoses yield other aldonic acids. ates—glucuronate, galacturonate, and so forth.
Oxidation of the carbon at the other end of the carbon In the synthesis and metabolism of carbohy-
chain—C-6 of glucose, galactose, or mannose—forms the drates, the intermediates are very often not the sugars
BOX 7–1 Blood Glucose Measurements in the Diagnosis and

MEDICINE
Treatment of Diabetes
Glucose is the principal fuel for the brain. When the A second enzyme, a peroxidase, catalyzes the reaction
amount of glucose reaching the brain is too low, the of the H2O2 with colorless compound to create a colored
consequences can be dire: lethargy, coma, permanent product, which is quantified with a simple photometer
brain damage, and death (see Fig. 23–24). Animals that reads out the blood glucose concentration.
have evolved complex hormonal mechanisms to ensure Because blood glucose levels change with the tim-
that the concentration of glucose in the blood remains ing of meals and exercise, single-time measurements do
high enough (about 5 mM) to satisfy the brain’s needs, not reflect the average blood glucose over hours and
but not too high, because elevated blood glucose can days, so dangerous increases may go undetected. The
also have serious physiological consequences. average glucose concentration can be assessed by look-
Individuals with insulin-dependent diabetes mel- ing at its effect on hemoglobin, the oxygen-carrying
litus do not produce sufficient insulin, the hormone protein in erythrocytes (p. 163). Transporters in the
that normally serves to reduce blood glucose concen- erythrocyte membrane equilibrate intracellular and
tration, and if the diabetes is untreated their blood plasma glucose concentrations, so hemoglobin is con-
glucose levels may rise to severalfold higher than stantly exposed to glucose at whatever concentration is
normal. These high glucose levels are believed to be present in the blood. A nonenzymatic reaction occurs
at least one cause of the serious long-term conse- between glucose and primary amino groups in hemoglo-
quences of untreated diabetes—kidney failure, car- bin (either the amino-terminal Val or the ´-amino
diovascular disease, blindness, and impaired wound groups of Lys residues) (Fig. 1). The rate of this pro-
healing—so one goal of therapy is to provide just cess is proportional to the concentration of glucose, so
enough insulin (by injection) to keep blood glucose the reaction can be used as the basis for estimating the
levels near normal. To maintain the correct balance average blood glucose level over weeks. The amount of
of exercise, diet, and insulin for the individual, blood glycated hemoglobin (GHB) present at any time reflects
glucose concentration needs to be measured several the average blood glucose concentration over the circu-
times a day, and the amount of insulin injected lating “lifetime” of the erythrocyte (about 120 days),
adjusted appropriately. although the concentration in the last two weeks is the
The concentrations of glucose in blood and urine most important in setting the level of GHB.
can be determined by a simple assay for reducing The extent of hemoglobin glycation (so named
sugar, such as Fehling’s reaction, which for many years to distinguish it from glycosylation, the enzymatic
was used as a diagnostic test for diabetes. Modern mea- transfer of glucose to a protein) is measured clini-
surements require just a drop of blood, added to a test cally by extracting hemoglobin from a small sample of
strip containing the enzyme glucose oxidase, which blood and separating GHB from unmodified hemoglo-
catalyzes the following reaction: bin electrophoretically, taking advantage of the
glucose oxidase charge difference resulting from modification of the
D-Glucose 1 O2
amino group(s). Normal GHB values are about 5% of
D-Glucono-␦-lactone 1 H2O2
total hemoglobin (corresponding to blood glucose of
themselves but their phosphorylated derivatives. Con- Monosaccharides Are Reducing Agents
densation of phosphoric acid with one of the hydroxyl Monosaccharides can be oxidized by relatively
groups of a sugar forms a phosphate ester, as in glucose mild oxidizing agents such as cupric (Cu2⫹) ion.
6-phosphate (Fig. 7–9), the first metabolite in the path- The carbonyl carbon is oxidized to a carboxyl group.
way by which most organisms oxidize glucose for Glucose and other sugars capable of reducing cupric ion
energy. Sugar phosphates are relatively stable at neu- are called reducing sugars. Cupric ion oxidizes glu-
tral pH and bear a negative charge. One effect of sugar cose and certain other sugars to a complex mixture of
phosphorylation within cells is to trap the sugar inside carboxylic acids. This is the basis of Fehling’s reaction,
the cell; most cells do not have plasma membrane a semi-quantitative test for the presence of reducing
transporters for phosphorylated sugars. Phosphoryla- sugar that for many years was used to detect and mea-
tion also activates sugars for subsequent chemical sure elevated glucose levels in people with diabetes
transformation. Several important phosphorylated mellitus. Today, more sensitive methods that involve an
derivatives of sugars are components of nucleotides immobilized enzyme on a test strip are used; they
(discussed in the next chapter). require only a single drop of blood (Box 7–1). ■
120 mg/100 mL). In people with untreated diabetes, heterogeneous mixture of AGEs, advanced glyca-
however, this value may be as high as 13%, indicating tion end products. These products can leave the
an average blood glucose level of about 300 mg/ erythrocyte and form covalent cross-links
100 mL—dangerously high. One criterion for success between proteins, interfering with normal pro-
in an individual program of insulin therapy (the tim- tein function (Fig. 1). The accumulation of rela-
ing, frequency, and amount of insulin injected) is tively high concentrations of AGEs in people with
maintaining GHB values at about 7%. diabetes may, by cross-linking critical proteins,
In the hemoglobin glycation reaction, the first cause the damage to the kidneys, retinas, and
step (formation of a Schiff base) is followed by a cardiovascular system that characterizes the dis-
series of rearrangements, oxidations, and dehy- ease. This pathogenic process is a potential
drations of the carbohydrate moiety to produce a target for drug action.
HOCH2
OH H
H H
C⫽O ⫹ H2N⫺R HOCH2
OH H
HO Hemoglobin OH
H H H
H OH CH2⫺N⫺R
Glucose OH
1 HO
HOCH2 O
H
OH H Ketoamine FIGURE 1 The nonenzymatic reac-
H H 3
tion of glucose with a primary
C⫽N⫺R
OH H HOCH2 O H amino group in hemoglobin begins
HO CH2⫺N⫺R
with 1 formation of a Schiff base,
H HO
H OH H OH which 2 undergoes a rearrange-
Schiff base ment to generate a stable product;
2a
HO H 3 this ketoamine can further
HOCH2 Glycated hemoglobin cyclize to yield GHB. 4 Subsequent
OH 4 (GHB)
reactions generate advanced glyca-
H H H tion end products (AGEs), such as
C⫺N⫺R AGEs
OH ´-N-carboxymethyllysine and meth-
HO 5 ylglyoxal, compounds that 5 can
Protein cross-linking damage other proteins by cross-
H OH
? ? ? linking them, causing pathological
2b
Damage to kidneys, retinas, cardiovascular system changes.
Disaccharides Contain a Glycosidic Bond carbon (one not involved in a glycosidic bond) is com-
monly called the reducing end.
Disaccharides (such as maltose, lactose, and sucrose)
The disaccharide maltose (Fig. 7–10) contains two
consist of two monosaccharides joined covalently by an
D-glucose residues joined by a glycosidic linkage
O-glycosidic bond, which is formed when a hydroxyl
between C-1 (the anomeric carbon) of one glucose
group of one sugar molecule, typically cyclic, reacts with
residue and C-4 of the other. Because the disaccharide
the anomeric carbon of the other (Fig. 7–10). This reac-
retains a free anomeric carbon (C-1 of the glucose resi-
tion represents the formation of an acetal from a hemiac-
due on the right in Fig. 7–10), maltose is a reducing
etal (such as glucopyranose) and an alcohol (a hydroxyl
sugar. The configuration of the anomeric carbon atom in
group of the second sugar molecule) (Fig. 7–5), and the
the glycosidic linkage is ␣. The glucose residue with the
resulting compound is called a glycoside. Glycosidic
free anomeric carbon is capable of existing in ␣- and
bonds are readily hydrolyzed by acid but resist cleavage
␤-pyranose forms.
by base. Thus disaccharides can be hydrolyzed to yield
their free monosaccharide components by boiling with
KEY CONVENTION: To name reducing disaccharides such
dilute acid. N-glycosyl bonds join the anomeric carbon
as maltose unambiguously, and especially to name more
of a sugar to a nitrogen atom in glycoproteins (see Fig.
complex oligosaccharides, several rules are followed. By
7–30) and nucleotides (see Fig. 8–1).
convention, the name describes the compound written
The oxidation of a sugar by cupric ion (the reaction
with its nonreducing end to the left, and we can “build
that defines a reducing sugar) occurs only with the linear
up” the name in the following order. (1) Give the con-
form, which exists in equilibrium with the cyclic form(s).
figuration (␣ or ␤) at the anomeric carbon joining the
When the anomeric carbon is involved in a glycosidic
first monosaccharide unit (on the left) to the second.
bond (that is, when the compound is a full acetal or ketal;
(2) Name the nonreducing residue; to distinguish five-
see Fig. 7–5), the easy interconversion of linear and
and six-membered ring structures, insert “furano” or
cyclic forms shown in Figure 7–6 is prevented. Because
“pyrano” into the name. (3) Indicate in parentheses
the carbonyl carbon can be oxidized only when the sugar
the two carbon atoms joined by the glycosidic bond,
is in its linear form, formation of a glycosidic bond ren-
with an arrow connecting the two numbers; for example,
ders a sugar nonreducing. In describing disaccharides or
(1S 4) shows that C-1 of the first-named sugar residue
polysaccharides, the end of a chain with a free anomeric
is joined to C-4 of the second. (4) Name the second
residue. If there is a third residue, describe the second
glycosidic bond by the same conventions. (To shorten
CH2OH CH2OH the description of complex polysaccharides, three-letter
O hemiacetal O abbreviations or colored symbols for the monosaccha-
H H H H H OH rides are often used, as given in Table 7–1.) Following
OH H
⫹
OH H this convention for naming oligosaccharides, maltose
HO OH HO H is ␣-D-glucopyranosyl-(1S4)-D-glucopyranose. Because
alcohol
H OH H OH
␣-D-Glucose ␤-D-Glucose
hydrolysis condensation
H2O H 2O
TABLE 7–1 Symbols and Abbreviations for
Common Monosaccharides and Some
6 CH 6 CH
2OH 2OH
5
acetal
5 of Their Derivatives
O O hemiacetal
H H H OH
H H Abequose Abe Glucuronic acid GlcA
4 1 4 1
OH H OH H Arabinose Ara Galactosamine GalN
HO H
O
3 2 3 2 Fructose Fru Glucosamine GlcN
H OH H OH Fucose Fuc N-Acetylgalactosamine GalNAc
Maltose
␣-D-glucopyranosyl-(1n4)-D-glucopyranose Galactose Gal N-Acetylglucosamine GlcNAc
Glucose Glc Iduronic acid IdoA
FIGURE 7–10 Formation of maltose. A disaccharide is formed from two
monosaccharides (here, two molecules of D-glucose) when an OOH Mannose Man Muramic acid Mur
(alcohol) of one monosaccharide molecule (right) condenses with the Rhamnose Rha N-Acetylmuramic acid Mur2Ac
intramolecular hemiacetal of the other (left), with elimination of H2O and Ribose Rib N-Acetylneuraminic
formation of a glycosidic bond. The reversal of this reaction is hydroly- acid (a sialic acid) Neu5Ac
Xylose Xyl
sis—attack by H2O on the glycosidic bond. The maltose molecule shown
here retains a reducing hemiacetal at the C-1 not involved in the glycosid- Note: In a commonly used convention, hexoses are represented as circles,
ic bond. Because mutarotation interconverts the ␣ and ␤ forms of the N-acetylhexosamines as squares, and hexosamines as squares divided diagonally. All
sugars with the “gluco” configuration are blue, those with the “galacto” configuration are
hemiacetal, the bonds at this position are sometimes depicted with wavy yellow, and “manno” sugars are green. Other substituents can be added as needed:
lines, as shown here, to indicate that the structure may be either ␣ or ␤. sulfate (S), phosphate (P), O-acetyl (OAc), or O-methyl (OMe).
most sugars encountered in this book are the D enantio- specifying the anomeric carbons and their configura-
mers and the pyranose form of hexoses predominates, tions. For example, the abbreviated name of sucrose is
we generally use a shortened version of the formal either Glc(␣142␤)Fru or Fru(␤241␣)Glc. Sucrose is
name of such compounds, giving the configuration of a major intermediate product of photosynthesis; in
the anomeric carbon and naming the carbons joined by many plants it is the principal form in which sugar is
the glycosidic bond. In this abbreviated nomenclature, transported from the leaves to other parts of the plant
maltose is Glc(␣1S 4)Glc. ■ body. Trehalose, Glc(␣141␣)Glc (Fig. 7–11)—a disac-
charide of D-glucose that, like sucrose, is a nonreducing
The disaccharide lactose (Fig. 7–11), which yields sugar—is a major constituent of the circulating fluid
D-galactose and D-glucose on hydrolysis, occurs natu- (hemolymph) of insects, serving as an energy-storage
rally in milk. The anomeric carbon of the glucose resi- compound. Lactose gives milk its sweetness, and
due is available for oxidation, and thus lactose is a sucrose, of course, is table sugar. Trehalose is also used
reducing disaccharide. Its abbreviated name is commercially as a sweetener. Box 7–2 explains how
Gal(␤1S 4)Glc. Sucrose (table sugar) is a disaccharide humans detect sweetness, and how artificial sweeteners
of glucose and fructose. It is formed by plants but not by such as aspartame act.
animals. In contrast to maltose and lactose, sucrose
contains no free anomeric carbon atom; the anomeric
carbons of both monosaccharide units are involved in SUMMARY 7.1 Monosaccharides and Disaccharides
the glycosidic bond (Fig. 7–11). Sucrose is therefore Sugars (also called saccharides) are compounds
a nonreducing sugar, and its stability toward oxidation containing an aldehyde or ketone group and two or
makes it a suitable molecule for the storage and trans- more hydroxyl groups.
port of energy in plants. In the abbreviated nomencla-
ture, a double-headed arrow connects the symbols Monosaccharides generally contain several chiral
carbons and therefore exist in a variety of
stereochemical forms, which may be represented
6 CH 6 CH
2OH 2OH on paper as Fischer projections. Epimers are
5 O 5
O sugars that differ in configuration at only one
HO H H H OH
4 1 ␤ O 4 1 ␤ carbon atom.
OH H OH H
H H H Monosaccharides commonly form internal
3 2 3 2 hemiacetals or hemiketals, in which the aldehyde
H OH H OH
or ketone group joins with a hydroxyl group of the
Lactose (␤ form)
␤-D-galactopyranosyl-(1n4)-␤-D-glucopyranose same molecule, creating a cyclic structure; this can
Gal(␤1n4)Glc be represented as a Haworth perspective formula.
The carbon atom originally found in the aldehyde
6 CH
2OH 1 or ketone group (the anomeric carbon) can
O HOCH2
H
5
H O H assume either of two configurations, a and ␤,
H
4 1 ␣ ␤ 2 5 which are interconvertible by mutarotation. In the
OH H H HO
HO CH2OH linear form of the monosaccharide, which is in
O
3 2 3 4
6
equilibrium with the cyclic forms, the anomeric
H OH OH H carbon is easily oxidized, making the compound a
Sucrose reducing sugar.
␤-D-fructofuranosyl ␣-D-glucopyranoside
Fru(2 ␤ n
n ␣1)Glc Glc(␣1n2
n ␤)Fru A hydroxyl group of one monosaccharide can
add to the anomeric carbon of a second
6 CH
2OH H monosaccharide to form an acetal called a
O O 5
H
5
H glycoside. In this disaccharide, the glycosidic
H HOCH2 OH
4 1 ␣ O ␣ 1 6 4 bond protects the anomeric carbon from
OH H OH H oxidation, making it a nonreducing sugar.
HO H H
3 2 2 3 Oligosaccharides are short polymers of several
H OH H OH
monosaccharides joined by glycosidic bonds.
Trehalose
␣-D-glucopyranosyl ␣-D-glucopyranoside At one end of the chain, the reducing end, is a
Glc(␣1n1
n ␣)Glc monosaccharide unit with its anomeric carbon
not involved in a glycosidic bond.
FIGURE 7–11 Two common disaccharides. Like maltose in Figure 7–10,
these are shown as Haworth perspectives. The common name, full sys- The common nomenclature for di- or
tematic name, and abbreviation are given for each disaccharide. Formal oligosaccharides specifies the order of
nomenclature for sucrose names glucose as the parent glycoside, monosaccharide units, the configuration at each
although it is typically depicted as shown, with glucose on the left. The anomeric carbon, and the carbon atoms involved in
two abbreviated symbols shown for sucrose are equivalent (;). the glycosidic linkage(s).
BOX 7–2 Sugar Is Sweet, and So Are . . . a Few Other Things

Sweetness is one of the five basic flavors that drates that are major fuels for most organisms. Most
humans can taste (Fig. 1); the others are sour, bit- simple sugars, including sucrose, glucose, and fructose,
ter, salty, and umami. Sweet taste is detected by
Alitame
protein receptors in the plasma membranes of gusta- Neotame Glucose
tory cells in the taste buds on the surface of the Aspartame Sucrose
tongue. In humans, two closely related genes (T1R2 Sucralose Fructose
and T1R3) encode sweetness receptors (Fig. 2).
When a molecule with a compatible structure binds
these receptors on a gustatory cell’s extracellular
domain, it triggers a series of events in the cell
(including activation of a GTP-binding protein; see T1R2 T1R3
Brazzein
Fig. 12–42) that lead to an electrical signal being
sent to the brain that is interpreted there as “sweet.” Brazzein
CRD
During evolution, there has probably been selection
for the ability to taste compounds found in foods
containing important nutrients, such as the carbohy-
GTP-binding protein GTP-binding protein

(inactive) (active)
FIGURE 2 The receptor for sweet-tasting substances, with its regions

of interaction (short arrows) with various sweet-tasting compounds
indicated. Each receptor has an extracellular domain, a cysteine-rich
domain (CRD), and a membrane domain with seven transmembrane
helices, a common feature of signaling receptors. Artificial sweeten-
ers bind to only one of the two receptor subunits; natural sugars bind
to both. See Chapter 1, Problem 14, for the structures of many of
FIGURE 1 A strong stimulus for the sweetness receptors. these artificial sweeteners.
7.2 Polysaccharides Homopolysaccharides Heteropolysaccharides
Most carbohydrates found in nature occur as polysac- Unbranched Branched Two Multiple
monomer monomer
charides, polymers of medium to high molecular weight types, types,
(Mr .20,000). Polysaccharides, also called glycans, dif- unbranched branched
fer from each other in the identity of their recurring
monosaccharide units, in the length of their chains, in
the types of bonds linking the units, and in the degree of
branching. Homopolysaccharides contain only a single
monomeric species; heteropolysaccharides contain
two or more different kinds (Fig. 7–12). Some homo-
polysaccharides serve as storage forms of monosaccha-
rides that are used as fuels; starch and glycogen are
homopolysaccharides of this type. Other homopolysac-
charides (cellulose and chitin, for example) serve as
structural elements in plant cell walls and animal exo-
skeletons. Heteropolysaccharides provide extracellular
support for organisms of all kingdoms. For example, the
rigid layer of the bacterial cell envelope (the peptidogly-
can) is composed in part of a heteropolysaccharide FIGURE 7–12 Homo- and heteropolysaccharides. Polysaccharides may
built from two alternating monosaccharide units (see be composed of one, two, or several different monosaccharides, in straight
Fig. 20–30). In animal tissues, the extracellular space is or branched chains of varying length.
7.2 Polysaccharides 255
taste sweet, but there are other classes of compounds gen. Site B⫺ contains a group with a partially nega-
that also bind the sweet receptors: the amino acids tive oxygen available to hydrogen-bond with some
glycine, alanine, and serine are mildly sweet and harmless; partially positive atom on the sweetener molecule,
nitrobenzene and ethylene glycol have a strong sweet such as the amine group of aspartame. Site X is ori-
taste, but are toxic. (See Box 18–2 for a remarkable ented perpendicular to the other two groups and is
medical mystery involving ethylene glycol poisoning.) capable of interacting with a hydrophobic patch on
Several natural products are extraordinarily sweet: the sweetener molecule, such as the benzene ring of
stevioside, a sugar derivative isolated from the leaves aspartame.
of the stevia plant (Stevia rebaudiana Berton), is sev- When the steric match is correct, as on the left
eral hundred times sweeter than an equivalent amount of in Figure 3, the sweet receptor is stimulated and
sucrose (table sugar), and the small (54 amino acids) the signal “sweet” is conducted to the brain. When
protein brazzein, isolated from berries of the Oubli the match is not correct, as on the right in Figure 3,
vine (Pentadiplandra brazzeana Baillon) in Gabon the sweet receptor is not stimulated; in fact, in this
and Cameroon, is 17,000 times as sweet as sucrose on case, another receptor (for bitterness) is stimulated
a molar basis. Presumably the sweet taste of the ber- by the “wrong” stereoisomer of aspartame. Stereo-
ries encourages their consumption by animals, which isomerism really matters!
then disperse the seeds geographically so new plants
may be established.
There is great interest in the development of arti-
ficial sweeteners as weight-reduction aids—com- Tastes sweet Tastes bitter D,L- or (R,S)-
pounds that give foods a sweet taste without adding Aspartame
T1R2
1R2
the calories found in sugars. The artificial sweetener
L,L- or (S,S)-
aspartame demonstrates the importance of stereo- Aspa
Aspartame
chemistry in biology (Fig. 3). According to one simple
model of sweetness receptor binding, binding involves
three sites on the receptor: AH⫹, B⫺, and X. Site AH⫹
contains some group (an alcohol or amine) that can X X
B⫺ B⫺
form a hydrogen bond with a partial negative charge, AH⫹ AH⫹
such as a carbonyl oxygen, on the sweetener molecule;
the carboxylic acid of aspartame contains such an oxy- FIGURE 3 Stereochemical basis for the taste of two isomers of aspartame.
occupied by several types of heteropolysaccharides, Starch and glycogen molecules are heavily hydrated,
which form a matrix that holds individual cells together because they have many exposed hydroxyl groups avail-
and provides protection, shape, and support to cells, able to hydrogen-bond with water. Most plant cells have
tissues, and organs. the ability to form starch (see Fig. 20–2), and starch stor-
Unlike proteins, polysaccharides generally do not age is especially abundant in tubers (underground
have defining molecular weights. This difference is a stems), such as potatoes, and in seeds.
consequence of the mechanisms of assembly of the two Starch contains two types of glucose polymer,
types of polymer. As we shall see in Chapter 27, pro- amylose and amylopectin (Fig. 7–13). Amylose con-
teins are synthesized on a template (messenger RNA) sists of long, unbranched chains of D-glucose residues
of defined sequence and length, by enzymes that follow connected by (␣1S 4) linkages (as in maltose). Such
the template exactly. For polysaccharide synthesis chains vary in molecular weight from a few thousand to
there is no template; rather, the program for polysac- more than a million. Amylopectin also has a high molec-
charide synthesis is intrinsic to the enzymes that cata- ular weight (up to 200 million) but unlike amylose is
lyze the polymerization of the monomeric units, and highly branched. The glycosidic linkages joining succes-
there is no specific stopping point in the synthetic pro- sive glucose residues in amylopectin chains are (␣1S 4);
cess; the products thus vary in length. the branch points (occurring every 24 to 30 residues)
are (␣1S 6) linkages.
Glycogen is the main storage polysaccharide of ani-
Some Homopolysaccharides Are Stored Forms of Fuel mal cells. Like amylopectin, glycogen is a polymer of
The most important storage polysaccharides are starch in (␣1S 4)-linked subunits of glucose, with (␣1S 6)-linked
plant cells and glycogen in animal cells. Both polysaccha- branches, but glycogen is more extensively branched (on
rides occur intracellularly as large clusters or granules. average, every 8 to 12 residues) and more compact than
6 CH
2OH CH2OH CH2OH CH2OH
5 O O O O
H H H H H H H H H H
Nonreducing H H Reducing
4 1 ␣ 4 1 ␣ 4 1 ␣ 4 1 ␣
end OH H OH H OH H OH H end
O O O O
3 2
H OH H OH H OH H OH
(a) Amylose
6 CH
2OH
O
H H H
4 1
OH H
O (␣1n6)
branch
Branch H OH point
O
A Amylopectin Amylose
6 CH
2
O
H H Reducing
H ends
4 1
OH H Nonreducing
O O ends
Main H OH (a1n6)
chain branch points
(b) (c)
FIGURE 7–13 Glycogen and starch. (a) A short segment of amylose, a amylopectin (black) form double-helical structures with each other or
linear polymer of D-glucose residues in (␣1S 4) linkage. A single chain with amylose strands (blue). Amylopectin has frequent (␣1S 6) branch
can contain several thousand glucose residues. Amylopectin has stretches points (red). Glucose residues at the nonreducing ends of the outer
of similarly linked residues between branch points. Glycogen has the same branches are removed enzymatically during the mobilization of starch for
basic structure, but has more branching than amylopectin. (b) An (␣1S 6) energy production. Glycogen has a similar structure but is more highly
branch point of glycogen or amylopectin. (c) A cluster of amylose and branched and more compact.
amylopectin like that believed to occur in starch granules. Strands of
starch. Glycogen is especially abundant in the liver, concentration of 0.4 M and an external concentration of
where it may constitute as much as 7% of the wet weight; about 5 mM (the concentration in the blood of a mam-
it is also present in skeletal muscle. In hepatocytes glyco- mal), the free-energy change for glucose uptake into
gen is found in large granules, which are themselves cells against this very high concentration gradient
clusters of smaller granules composed of single, highly would be prohibitively large.
branched glycogen molecules with an average molecular Dextrans are bacterial and yeast polysaccharides
weight of several million. Such glycogen granules also made up of (␣1S 6)-linked poly-D-glucose; all have
contain, in tightly bound form, the enzymes responsible (␣1S 3) branches, and some also have (␣1S 2) or
for the synthesis and degradation of glycogen. (␣1S 4) branches. Dental plaque, formed by bacteria
Because each branch in glycogen ends with a nonre- growing on the surface of teeth, is rich in dextrans,
ducing sugar unit, a glycogen molecule with n branches which are adhesive and allow the bacteria to stick to
has n 1 1 nonreducing ends, but only one reducing end. teeth and to each other. Dextrans also provide a source
When glycogen is used as an energy source, glucose units of glucose for bacterial metabolism. Synthetic dextrans
are removed one at a time from the nonreducing ends. are used in several commercial products (for example,
Degradative enzymes that act only at nonreducing ends Sephadex) that serve in the fractionation of proteins by
can work simultaneously on the many branches, speed- size-exclusion chromatography (see Fig. 3–17b). The
ing the conversion of the polymer to monosaccharides. dextrans in these products are chemically cross-linked
Why not store glucose in its monomeric form? It has to form insoluble materials of various sizes.
been calculated that hepatocytes store glycogen equiva-
lent to a glucose concentration of 0.4 M. The actual
concentration of glycogen, which is insoluble and con- Some Homopolysaccharides Serve Structural Roles
tributes little to the osmolarity of the cytosol, is about Cellulose, a fibrous, tough, water-insoluble substance,
0.01 ␮M. If the cytosol contained 0.4 M glucose, the is found in the cell walls of plants, particularly in stalks,
osmolarity would be threateningly elevated, leading to stems, trunks, and all the woody portions of the plant
osmotic entry of water that might rupture the cell (see body. Cellulose constitutes much of the mass of wood,
Fig. 2–13). Furthermore, with an intracellular glucose and cotton is almost pure cellulose. Like amylose, the
OH OH genetic studies have revealed that genes encoding

HO 6 O cellulose-degrading enzymes are present in the genomes
4
O
O
5 2
O
of a wide range of invertebrate animals, including arthro-
O HO pods and nematodes. There is one important exception to
OH 1
OH
3
the absence of cellulase in vertebrates: ruminant animals
(␤1n4)-linked D-glucose units such as cattle, sheep, and goats harbor symbiotic microor-
FIGURE 7–14 Cellulose. Two units of a cellulose chain; the D-glucose
ganisms in the rumen (the first of their four stomach
residues are in (␤1S 4) linkage. The rigid chair structures can rotate rela-
compartments) that can hydrolyze cellulose, allowing the
tive to one another.
animal to degrade dietary cellulose from soft grasses, but
not from woody plants. Fermentation in the rumen yields
cellulose molecule is a linear, unbranched homopolysac- acetate, propionate, and ␤-hydroxybutyrate, which the
charide, consisting of 10,000 to 15,000 D-glucose units. animal uses to synthesize the sugars in milk (p. 560).
But there is a very important difference: in cellulose the Biomass (such as switch grass) that is rich in cel-
glucose residues have the ␤ configuration (Fig. 7–14), lulose can be used as starting material for the fermenta-
whereas in amylose the glucose is in the ␣ configuration of carbohydrates to ethanol, to be used as a gasoline
tion. The glucose residues in cellulose are linked by additive. The annual production of biomass on Earth
(␤1S4) glycosidic bonds, in contrast to the (␣1S 4) (accomplished primarily by photosynthetic organisms)
bonds of amylose. This difference causes individual mol- is the energetic equivalent of nearly a trillion barrels of
ecules of cellulose and amylose to fold differently in crude oil, when converted to ethanol by fermentation.
space, giving them very different macroscopic struc- Because of their potential use in biomass conversion to
tures and physical properties (see below). The tough, bioenergy, cellulose-degrading enzymes such as cellu-
fibrous nature of cellulose makes it useful in such com- lase are under vigorous investigation. Supramolecular
mercial products as cardboard and insulation material, complexes called cellulosomes, found on the outside
and it is a major constituent of cotton and linen fabrics. surface of the bacterium Clostridium cellulolyticum,
Cellulose is also the starting material for the commercial include the catalytic subunit of cellulase, along with
production of cellophane and rayon. proteins that hold one or more cellulase molecules to
Glycogen and starch ingested in the diet are hydro- the bacterial surface, and a subunit that binds cellulose
lyzed by ␣-amylases and glycosidases, enzymes in saliva and positions it in the catalytic site.
and the intestine that break (␣1S 4) glycosidic bonds A major fraction of photosynthetic biomass is the
between glucose units. Most vertebrate animals cannot woody portion of plants and trees, which consists of cel-
use cellulose as a fuel source, because they lack an lulose plus several other polymers derived from carbo-
enzyme to hydrolyze the (␤1S4) linkages. Termites hydrates that are not easily digestible, either chemically
readily digest cellulose (and therefore wood), but only or biologically. Lignins, for example, make up some 30%
because their intestinal tract harbors a symbiotic micro- of the mass of wood. Synthesized from precursors that
organism, Trichonympha, that secretes cellulase, which include phenylalanine and glucose, lignins are complex
hydrolyzes the (␤1S4) linkages (Fig. 7–15). Molecular polymers with covalent cross-links to cellulose that
complicate the digestion of cellulose by cellulase. If
woody plants are to be used in the production of ethanol
from biomass, better means of digesting wood compo-
nents will need to be found.
Chitin is a linear homopolysaccharide composed
of N-acetylglucosamine residues in (␤1S4) linkage
(Fig. 7–16). The only chemical difference from cellulose
is the replacement of the hydroxyl group at C-2 with an
acetylated amino group. Chitin forms extended fibers
similar to those of cellulose, and like cellulose cannot be
digested by vertebrates. Chitin is the principal component
of the hard exoskeletons of nearly a million species of
arthropods—insects, lobsters, and crabs, for example—
and is probably the second most abundant polysaccha-
FIGURE 7–15 Cellulose breakdown by Trichonympha, a protist in the gut ride, next to cellulose, in nature; an estimated 1 billion
of a wood-eating termite. Trichonympha produces the enzyme cellulase, tons of chitin are produced each year in the biosphere.
which breaks the (␤1S4) glycosidic bonds in cellulose, making wood a
source of metabolizable sugar (glucose) for the protist and the termite. A Steric Factors and Hydrogen Bonding Influence
number of invertebrates can digest cellulose, but only a few vertebrates
(the ruminants, such as cattle, sheep, and goats); the ruminants are able
Homopolysaccharide Folding
to use cellulose as food because the first of their four stomach compart- The folding of polysaccharides in three dimensions fol-
ments (rumen) teems with bacteria and protists that secrete cellulase. lows the same principles as those governing polypeptide
CH3 CH3
A A
CPO CPO
A A
6 CH
2OH H NH CH2OH H NH
3 2
5
O O O O
H H OH H H H H OH H H
4 1 4 1
OH H H OH H H
O H H O H H
5 O O
3 2
H NH CH2OH H NH CH2OH
A 6 A
CPO CPO
A A
(a) CH3 CH3
FIGURE 7–16 Chitin. (a) A short segment of chitin, a homopoly-

mer of N-acetyl-D-glucosamine units in (␤1S4) linkage. (b) A
spotted June beetle (Pelidnota punctata), showing its surface
(b) armor (exoskeleton) of chitin.
structure: subunits with a more-or-less rigid structure thus determination of the structure by x-ray diffraction.
dictated by covalent bonds form three-dimensional The average plane of each residue along the amylose
macromolecular structures that are stabilized by weak chain forms a 608 angle with the average plane of the
interactions within or between molecules, such as
hydrogen bonds and hydrophobic and van der Waals CH2OH O H O OH
interactions, and, for polymers with charged subunits, O 4
␾ ␺
1
electrostatic interactions. Because polysaccharides O 4

1
have so many hydroxyl groups, hydrogen bonding has HO HO CH2OH
O
an especially important influence on their structure. Cellulose
Glycogen, starch, and cellulose are composed of pyran- (␤1 4)Glc repeats
oside subunits (having six-membered rings), as are the
O
oligosaccharides of glycoproteins and glycolipids, to be 1 ␾ ␺ 4
CH2OH CH2OH O
discussed later. Such molecules can be represented as a
O
series of rigid pyranose rings connected by an oxygen 4
atom bridging two carbon atoms (the glycosidic bond). O O 1

H O HO
There is, in principle, free rotation about both COO HO
bonds linking the residues (Fig. 7–14), but as in poly- H
peptides (see Figs 4–2, 4–9), rotation about each bond Amylose
is limited by steric hindrance by substituents. The (␣1 4)Glc repeats
three-dimensional structures of these molecules can be
described in terms of the dihedral angles, ␾ and ␺, O O ␾
about the glycosidic bond (Fig. 7–17), analogous to 6
CH2
1 ␺
O
␻
angles ␾ and ␺ made by the peptide bond (see Fig. 4–2). O C 1
OH 6
The bulkiness of the pyranose ring and its substitu-
HO 5
ents, and electronic effects at the anomeric carbon, HO
HO
place constraints on the angles ␾ and ␺; thus certain HO
conformations are much more stable than others, as can HO
be shown on a map of energy as a function of ␾ and ␺ Dextran
(Fig. 7–18). (␣1 6)Glc repeats (with (␣1 3) branches, not shown)
The most stable three-dimensional structure for the FIGURE 7–17 Conformation at the glycosidic bonds of cellulose, amy-
(␣1S 4)-linked chains of starch and glycogen is a tightly lose, and dextran. The polymers are depicted as rigid pyranose rings
coiled helix (Fig. 7–19), stabilized by interchain joined by glycosidic bonds, with free rotation about these bonds. Note
hydrogen bonds. In amylose (with no branches) this that in dextran there is also free rotation about the bond between C-5 and
structure is regular enough to allow crystallization and C-6 (torsion angle ␻ (omega)).
䊉 ␾,␺ ⫽ 30,40
(a) (b) 䊉 ␾,␺ 170, 170

FIGURE 7–18 A map of favored conformations for oligosaccharides and the result is a map of preferred conformations. This is analogous to the
polysaccharides. The torsion angles ␺ and ␾ (see Fig. 7–17), which de- Ramachandran plot for peptides (see Figs 4–3, 4–9). (b) Two energetic
fine the spatial relationship between adjacent rings, can in principle have extremes for the disaccharide Gal(␤1S 3)Gal; these values fall on the
any value from 08 to 3608. In fact, some of the torsion angles would give energy diagram (a) as shown by the red and blue dots. The red dot
conformations that are sterically hindered, whereas others give conforma- indicates the least favored conformation; the blue dot, the most favored
tions that maximize hydrogen bonding. (a) When the relative energy (兺) conformation. The known conformations of the three polysaccharides
is plotted for each value of ␾ and ␺, with isoenergy (“same energy”) con- shown in Figure 7–17 have been determined by x-ray crystallography, and
tours drawn at intervals of 1 kcal/mol above the minimum energy state, all fall within the lowest-energy regions of the map.
preceding residue, so the helical structure has six resi- complex. This interaction is a common qualitative test
dues per turn. For amylose, the core of the helix is of for amylose.
precisely the right dimensions to accommodate iodine For cellulose, the most stable conformation is that
as complex ions (I3⫺ and I5⫺), giving an intensely blue in which each chair is turned 1808 relative to its neigh-
bors, yielding a straight, extended chain. All —OH
groups are available for hydrogen bonding with neigh-
HO OH
O boring chains. With several chains lying side by side, a
O stabilizing network of interchain and intrachain hydro-
CH2OH gen bonds produces straight, stable supramolecular
O fibers of great tensile strength (Fig. 7–20). This prop-
erty of cellulose has made it a useful substance to civili-
CH2OH
zations for millennia. Many manufactured products,
HO including papyrus, paper, cardboard, rayon, insulating
tiles, and a variety of other useful materials, are derived
from cellulose. The water content of these materials is
O low because extensive interchain hydrogen bonding
HO
between cellulose molecules satisfies their capacity for
hydrogen-bond formation.
(␣1n4)-linked
D-glucose units Bacterial and Algal Cell Walls Contain Structural
(a) (b) Heteropolysaccharides
FIGURE 7–19 Helical structure of starch (amylose). (a) In the most The rigid component of bacterial cell walls (peptidogly-
stable conformation, with adjacent rigid chairs, the polysaccharide chain can) is a heteropolymer of alternating (␤1S 4)-linked
is curved, rather than linear as in cellulose (see Fig. 7–14). (b) A model of N-acetylglucosamine and N-acetylmuramic acid resi-
a segment of amylose; for clarity, the hydroxyl groups have been omit- dues (see Fig. 20–30). The linear polymers lie side by
ted from all but one of the glucose residues. Compare the two residues side in the cell wall, cross-linked by short peptides, the
shaded in pink with the chemical structures in (a). The conformation of exact structure of which depends on the bacterial spe-
(a1S4) linkages in amylose, amylopectin, and glycogen causes these cies. The peptide cross-links weld the polysaccharide
polymers to assume tightly coiled helical structures. These compact chains into a strong sheath (peptidoglycan) that envel-
structures produce the dense granules of stored starch or glycogen seen ops the entire cell and prevents cellular swelling and
in many cells (see Fig. 20–2). lysis due to the osmotic entry of water. The enzyme
O
6 HO 6
6
CH2 1
4 CH2OH O 3 O
2
5 1 5 2
OSO⫺
3
4 O 5
OH 4
O
2 3 1
3
Agarose
3)D-Gal(1 4)3,6-anhydro-L-Gal2S (1
repeating units
FIGURE 7–21 Agarose. The repeating unit consists of D-galactose
(␤1S4)-linked to 3,6-anhydro-L-galactose (in which an ether bridge
connects C-3 and C-6). These units are joined by (␣1S3) glycosidic
links to form a polymer 600 to 700 residues long. A small fraction of
the 3,6-anhydrogalactose residues have a sulfate ester at C-2 (as shown
here). The open parentheses in the systematic name indicate that the
repeating unit extends from both ends.
FIGURE 7–20 Cellulose chains. Scale drawing of segments of two parallel
cellulose chains, showing the conformation of the D-glucose residues Glycosaminoglycans Are Heteropolysaccharides
and the hydrogen-bond cross-links. In the hexose unit at the lower left,
all hydrogen atoms are shown; in the other three hexose units, the
of the Extracellular Matrix
hydrogens attached to carbon have been omitted for clarity, as they do The extracellular space in the tissues of multicellular
not participate in hydrogen bonding. animals is filled with a gel-like material, the extracel-
lular matrix (ECM), also called ground substance,
which holds the cells together and provides a porous
lysozyme kills bacteria by hydrolyzing the (␤1S4) pathway for the diffusion of nutrients and oxygen to
glycosidic bond between N-acetylglucosamine and individual cells. The ECM that surrounds fibroblasts and
N-acetylmuramic acid (see Fig. 6–27); the enzyme is other connective tissue cells is composed of an inter-
found in human tears, where it is presumably a defense locking meshwork of heteropolysaccharides and fibrous
against bacterial infections of the eye, and is also pro- proteins such as fibrillar collagens, elastins, and fibro-
duced by certain bacterial viruses to ensure their nectins. Basement membrane is a specialized ECM that
release from the host bacterial cell, an essential step of underlies epithelial cells; it consists of specialized col-
the viral infection cycle. Penicillin and related antibiot- lagens, laminins, and heteropolysaccharides. These
ics kill bacteria by preventing synthesis of the cross- heteropolysaccharides, the glycosaminoglycans, are a
links, leaving the cell wall too weak to resist osmotic family of linear polymers composed of repeating disac-
lysis (p. 224). charide units (Fig. 7–22). They are unique to animals
Certain marine red algae, including some of the and bacteria and are not found in plants. One of the two
seaweeds, have cell walls that contain agar, a mixture monosaccharides is always either N-acetylglucosamine
of sulfated heteropolysaccharides made up of D-galac- or N-acetylgalactosamine; the other is in most cases a
tose and an L-galactose derivative ether-linked between uronic acid, usually D-glucuronic or L-iduronic acid.
C-3 and C-6. Agar is a complex mixture of polysaccha- Some glycosaminoglycans contain esterified sulfate
rides, all with the same backbone structure but substi- groups. The combination of sulfate groups and the car-
tuted to varying degrees with sulfate and pyruvate. boxylate groups of the uronic acid residues gives glycos-
Agarose (Mr ⬃150,000) is the agar component with aminoglycans a very high density of negative charge. To
the fewest charged groups (sulfates, pyruvates) (Fig. minimize the repulsive forces among neighboring
7–21). The remarkable gel-forming property of agarose charged groups, these molecules assume an extended
makes it useful in the biochemistry laboratory. When a conformation in solution, forming a rodlike helix in
suspension of agarose in water is heated and cooled, which the negatively charged carboxylate groups occur
the agarose forms a double helix: two molecules in par- on alternate sides of the helix (as shown for heparin in
allel orientation twist together with a helix repeat of Fig. 7–22). The extended rod form also provides maxi-
three residues; water molecules are trapped in the cen- mum separation between the negatively charged sulfate
tral cavity. These structures in turn associate with each groups. The specific patterns of sulfated and nonsul-
other to form a gel—a three-dimensional matrix that fated sugar residues in glycosaminoglycans provide for
traps large amounts of water. Agarose gels are used as specific recognition by a variety of protein ligands that
inert supports for the electrophoretic separation of bind electrostatically to these molecules. The sulfated
nucleic acids, an essential part of the DNA-sequencing glycosaminoglycans are attached to extracellular pro-
process (p. 302). Agar is also used to form a surface for teins to form proteoglycans (Section 7.3).
the growth of bacterial colonies. Another commercial The glycosaminoglycan hyaluronan (hyaluronic
use of agar is for the capsules in which some vitamins acid) contains alternating residues of D-glucuronic acid
and drugs are packaged; the dried agar material dis- and N-acetylglucosamine (Fig. 7–22). With up to 50,000
solves readily in the stomach and is metabolically inert. repeats of the basic disaccharide unit, hyaluronan has a
Glycosaminoglycan Repeating disaccharide FIGURE 7–22 Repeating units of some common glycosaminoglycans of
Number of extracellular matrix. The molecules are copolymers of alternating uronic
disaccharides CH2OH acid and amino sugar residues (keratan sulfate is the exception), with
per chain O sulfate esters in any of several positions, except in hyaluronan. The ion-
H H O ized carboxylate and sulfate groups (red in the perspective formulas)
COO⫺ H
HO give these polymers their characteristic high negative charge. Therapeu-
O H
Hyaluronan H O (1n4) tic heparin contains primarily iduronic acid (IdoA) and a smaller propor-
H
⬃50,000 H NH tion of glucuronic acid (GlcA, not shown), and is generally highly sulfated
OH H A
H CP O and heterogeneous in length. The space-filling model shows a heparin
(1n3) A segment as its solution structure, as determined by NMR spectroscopy
H OH CH3
(PDB ID 1HPN). The carbons in the iduronic acid sulfate are colored
GlcA GlcNAc blue; those in glucosamine sulfate are green. Oxygen and sulfur atoms
are shown in their standard colors of red and yellow, respectively. The
CH2OH
hydrogen atoms are not shown (for clarity). Heparan sulfate (not
O
⫺
O3SO H shown) is similar to heparin but has a higher proportion of GlcA and
O
COO ⫺ fewer sulfate groups, arranged in a less regular pattern.
H
O H H
Chondroitin H O (1n4)
4-sulfate H some pathogenic bacteria, can hydrolyze the glycosidic
H NH
20–60 OH H A linkages of hyaluronan, rendering tissues more suscepti-
H CP O
(1n3) A ble to bacterial invasion. In many animal species, a similar
H OH CH3 enzyme in sperm hydrolyzes an outer glycosaminoglycan
GlcA GalNAc4S coat around the ovum, allowing sperm penetration.
Other glycosaminoglycans differ from hyaluronan in
CH2OSO⫺
3
O
three respects: they are generally much shorter poly-
CH2OH
H H
O mers, they are covalently linked to specific proteins
O
Keratan HO OH H (proteoglycans), and one or both monomeric units differ
H
sulfate O H from those of hyaluronan. Chondroitin sulfate (Greek
H (1n3)
⬃25 H H H NH
chondros, “cartilage”) contributes to the tensile strength
(1n4) A of cartilage, tendons, ligaments, and the walls of the
H OH CP O
A aorta. Dermatan sulfate (Greek derma, “skin”) contrib-
CH3 utes to the pliability of skin and is also present in blood
Gal GlcNAc6S vessels and heart valves. In this polymer, many of the
glucuronate residues present in chondroitin sulfate are
CH2OSO⫺3
replaced by their 5-epimer, L-iduronate (IdoA).
H O
H H H
O H
H (1n4) H COO⫺
COO⫺ OSO⫺
3
Heparin O O O O
OH H H OH OH
15–90 H COO⫺ H
H NH SO⫺
3
(1n4) OH H OH H
H OSO⫺
3 HO H HO H
IdoA2S GlcNS3S6S
H OH H OH
-L-Iduronate -D-Glucuronate
(IdoA) (GlcA)
Keratan sulfates (Greek keras, “horn”) have no

uronic acid and their sulfate content is variable. They are
Heparin segment present in cornea, cartilage, bone, and a variety of horny
structures formed of dead cells: horn, hair, hoofs, nails,
and claws. Heparan sulfate (Greek hēpar, “liver”; it
molecular weight of several million; it forms clear, highly was originally isolated from dog liver) is produced by all
viscous solutions that serve as lubricants in the synovial animal cells and contains variable arrangements of sul-
fluid of joints and give the vitreous humor of the verte- fated and nonsulfated sugars. The sulfated segments of
brate eye its jellylike consistency (the Greek hyalos the chain allow it to interact with a large number of pro-
means “glass”; hyaluronan can have a glassy or translu- teins, including growth factors and ECM components, as
cent appearance). Hyaluronan is also a component of the well as various enzymes and factors present in plasma.
extracellular matrix of cartilage and tendons, to which it Heparin is a fractionated form of heparan sulfate derived
contributes tensile strength and elasticity as a result of its mostly from mast cells (a type of leukocyte). Heparin is a
strong noncovalent interactions with other components therapeutic agent used to inhibit coagulation through its
of the matrix. Hyaluronidase, an enzyme secreted by capacity to bind the protease inhibitor antithrombin.
FIGURE 7–23 Interaction between a glycosaminoglycan and its binding pro-

tein. Fibroblast growth factor 1 (FGF1), its cell surface receptor (FGFR), and a
short segment of a glycosaminoglycan (heparin) were co-crystallized to yield
the structure shown here (PDB ID 1E0O). The proteins are represented as
surface contour images, with color to represent surface electrostatic poten-
tial: red, predominantly negative charge; blue, predominantly positive charge.
Heparin is shown in a ball-and-stick representation, with the negative charges
(OSO2 3 and OCOO ) attracted to the positive (blue) surface of the FGF1
2
protein. Heparin was used in this experiment, but the glycosaminoglycan

Glycosaminoglycan that binds FGF1 in vivo is heparan sulfate on the cell surface.
(heparin)
Heparin binding causes antithrombin to bind to and

inhibit thrombin, a protease essential to blood clotting.
The interaction is strongly electrostatic; heparin has the
highest negative charge density of any known biological
macromolecule (Fig. 7–23). Purified heparin is routinely
added to blood samples obtained for clinical analysis, and
to blood donated for transfusion, to prevent clotting.
Table 7–2 summarizes the composition, properties,
roles, and occurrence of the polysaccharides described
in Section 7.2.
TABLE 7–2 Structures and Roles of Some Polysaccharides

Size (number of
monosaccharide
Polymer Type* Repeating unit† units) Roles/significance
Starch Energy storage: in plants
Amylose Homo- (␣1S4)Glc, linear 50–5,000
Amylopectin Homo- (␣1S4)Glc, with Up to 106
(␣1S6)Glc
branches every
24–30 residues
Glycogen Homo- (␣1S4)Glc, with Up to 50,000 Energy storage: in bacteria and
(␣1S6)Glc animal cells
branches every
8–12 residues
Cellulose Homo- (␤1S4)Glc Up to 15,000 Structural: in plants, gives rigidity
and strength to cell walls
Chitin Homo- ( ␤1S4)GlcNAc Very large Structural: in insects, spiders,
crustaceans, gives rigidity and
strength to exoskeletons
Dextran Homo- (␣1S6)Glc, with Wide range Structural: in bacteria, extracellular
(␣1S3) branches adhesive
Peptidoglycan Hetero-; 4)Mur2Ac(␤1S4) Very large Structural: in bacteria, gives rigidity
peptides GlcNAc(␤1 and strength to cell envelope
attached
Agarose Hetero- 3)D-Gal(␤1S4)3,6- 1,000 Structural: in algae, cell wall material
anhydro-L-Gal(␣1
Hyaluronan (a Hetero-; 4)GlcA(␤1S3) Up to 100,000 Structural: in vertebrates, extracellular
glycosamino- acidic GlcNAc( ␤1 matrix of skin and connective tissue;
glycan) viscosity and lubrication in joints
*Each polymer is classified as a homopolysaccharide (homo-) or heteropolysaccharide (hetero-).
†
The abbreviated names for the peptidoglycan, agarose, and hyaluronan repeating units indicate that the polymer contains repeats of this disaccharide unit.
For example, in peptidoglycan, the GlcNAc of one disaccharide unit is (␤1S4)-linked to the first residue of the next disaccharide unit.
7.3 Glycoconjugates: Proteoglycans, Glycoproteins, and Glycosphingolipids 263
SUMMARY 7.2 Polysaccharides when the protein is malformed or superfluous; and oth-
ers serve as recognition sites for extracellular signal
Polysaccharides (glycans) serve as stored fuel and
molecules (growth factors, for example) or extracellu-
as structural components of cell walls and
lar parasites (bacteria or viruses). On almost every
extracellular matrix.
eukaryotic cell, specific oligosaccharide chains attached
The homopolysaccharides starch and glycogen are to components of the plasma membrane form a carbo-
stored fuels in plant, animal, and bacterial cells. hydrate layer (the glycocalyx), several nanometers
They consist of D-glucose with (␣1S4) linkages, thick, that serves as an information-rich surface that the
and both contain some branches. cell shows to its surroundings. These oligosaccharides
The homopolysaccharides cellulose, chitin, and are central players in cell-cell recognition and adhesion,
dextran serve structural roles. Cellulose, composed cell migration during development, blood clotting, the
of (␤1S4)-linked D-glucose residues, lends immune response, wound healing, and other cellular
strength and rigidity to plant cell walls. Chitin, a processes. In most of these cases, the informational
polymer of (␤1S4)-linked N-acetylglucosamine, carbohydrate is covalently joined to a protein or a lipid
strengthens the exoskeletons of arthropods. to form a glycoconjugate, which is the biologically
Dextran forms an adhesive coat around certain active molecule (Fig. 7–24).
bacteria. Proteoglycans are macromolecules of the cell sur-
face or extracellular matrix in which one or more sul-
Homopolysaccharides fold in three dimensions.
fated glycosaminoglycan chains are joined covalently to
The chair form of the pyranose ring is essentially
a membrane protein or a secreted protein. The glycos-
rigid, so the conformation of the polymers is
aminoglycan chain can bind to extracellular proteins
determined by rotation about the bonds from the
through electrostatic interactions between the protein
rings to the oxygen atom in the glycosidic linkage.
and the negatively charged sugar moieties on the pro-
Starch and glycogen form helical structures with
teoglycan. Proteoglycans are major components of all
intrachain hydrogen bonding; cellulose and chitin
extracellular matrices.
form long, straight strands that interact with
Glycoproteins have one or several oligosaccha-
neighboring strands.
rides of varying complexity joined covalently to a pro-
Bacterial and algal cell walls are strengthened by tein. They are usually found on the outer face of the
heteropolysaccharides—peptidoglycan in bacteria, plasma membrane (as part of the glycocalyx), in the
agar in red algae. The repeating disaccharide in extracellular matrix, and in the blood. Inside cells they are
peptidoglycan is GlcNAc(␤1S4)Mur2Ac; in agar, found in specific organelles such as Golgi complexes,
it is D-Gal(␤1S4)3,6-anhydro-L-Gal.
Proteoglycans
Glycosaminoglycans are extracellular +NH
3
heteropolysaccharides in which one of the two |
monosaccharide units is a uronic acid (keratin Chondroitin sulfate
sulfate is an exception) and the other an Ser
Heparan sulfate
N-acetylated amino sugar. Sulfate esters on some Ser Fuc
of the hydroxyl groups and on the amino group of Gal
some glucosamine residues in heparin and in Glycoproteins
Glc
Man
heparan sulfate give these polymers a high density +NH
3 Xylose
of negative charge, forcing them to assume |
GlcA
N-glycan
extended conformations. These polymers Asn GalNAc
(hyaluronan, chondroitin sulfate, dermatan GlcNAc
sulfate, and keratan sulfate) provide viscosity, Asn IdoA
adhesiveness, and tensile strength to the Neu5Ac
extracellular matrix. Glycosphingolipids
Ser/Thr
O-glycan
7.3 Glycoconjugates: Proteoglycans, Ser/Thr
Glycoproteins, and Glycosphingolipids Outside
In addition to their important roles as stored fuels

Membrane
(starch, glycogen, dextran) and as structural materials
(cellulose, chitin, peptidoglycans), polysaccharides and
oligosaccharides are information carriers. Some provide Inside | |
COO– COO–
communication between cells and their extracellular
surroundings; others label proteins for transport to and FIGURE 7–24 Glycoconjugates. The structures of some typical proteo-
localization in specific organelles, or for destruction glycans, glycoproteins, and glycosphingolipids described in the text.
PART II
BIOENERGETICS AND METABOLISM

13 Bioenergetics and Biochemical Reaction 19 Oxidative Phosphorylation and
Types 505 Photophosphorylation 731
14 Glycolysis, Gluconeogenesis, and the 20 Carbohydrate Biosynthesis in Plants and
Pentose Phosphate Pathway 543 Bacteria 799
15 Principles of Metabolic Regulation 587 21 Lipid Biosynthesis 833
16 The Citric Acid Cycle 633 22 Biosynthesis of Amino Acids, Nucleotides,
17 Fatty Acid Catabolism 667 and Related Molecules 881
18 Amino Acid Oxidation and the Production 23 Hormonal Regulation and Integration of
of Urea 695 Mammalian Metabolism 929
M
etabolism is a highly coordinated cellular activity their sole source of carbon, from which they construct
in which many multienzyme systems (metabolic all their carbon-containing biomolecules (see Fig. 1–5).
pathways) cooperate to (1) obtain chemical energy Some autotrophic organisms, such as cyanobacteria,
by capturing solar energy or degrading energy-rich can also use atmospheric nitrogen to generate all their
nutrients from the environment; (2) convert nutrient nitrogenous components. Heterotrophs cannot use
molecules into the cell’s own characteristic molecules, atmospheric carbon dioxide and must obtain carbon
including precursors of macromolecules; (3) polymerize from their environment in the form of relatively com-
monomeric precursors into macromolecules: proteins, plex organic molecules such as glucose. Multicellular
nucleic acids, and polysaccharides; and (4) synthesize animals and most microorganisms are heterotrophic.
and degrade biomolecules required for specialized cel- Autotrophic cells and organisms are relatively self-suffi-
lular functions, such as membrane lipids, intracellular cient, whereas heterotrophic cells and organisms, with
messengers, and pigments. their requirements for carbon in more complex forms,
Although metabolism embraces hundreds of differ- must subsist on the products of other organisms.
ent enzyme-catalyzed reactions, our major concern in Many autotrophic organisms are photosynthetic
Part II is the central metabolic pathways, which are few and obtain their energy from sunlight, whereas hetero-
in number and remarkably similar in all forms of life. trophic organisms obtain their energy from the degrada-
Living organisms can be divided into two large groups tion of organic nutrients produced by autotrophs. In our
according to the chemical form in which they obtain biosphere, autotrophs and heterotrophs live together
carbon from the environment. Autotrophs (such as in a vast, interdependent cycle in which autotrophic
photosynthetic bacteria, green algae, and vascular organisms use atmospheric carbon dioxide to build
plants) can use carbon dioxide from the atmosphere as their organic biomolecules, some of them generating
501
502 Bioenergetics and Metabolism
oxygen from water in the process. Heterotrophs in turn N2 in Denitrifying

Nitrogen-fixing
use the organic products of autotrophs as nutrients and bacteria and
atmosphere bacteria, archaea,
and fungi
return carbon dioxide to the atmosphere. Some of the archaea
oxidation reactions that produce carbon dioxide also

consume oxygen, converting it to water. Thus carbon, ⫹
NH4 NO⫺ 3
oxygen, and water are constantly cycled between the Ammonia Anammox Nitrate
bacteria
heterotrophic and autotrophic worlds, with solar energy Nitrifying
as the driving force for this global process (Fig. 1). bacteria
All living organisms also require a source of nitro- Nitrifying bacteria

and archaea NO⫺ 2
gen, which is necessary for the synthesis of amino acids, Nitrite
nucleotides, and other compounds. Bacteria and plants Plants
Animals
can generally use either ammonia or nitrate as their sole
Amino Plants
source of nitrogen, but vertebrates must obtain nitrogen acids
in the form of amino acids or other organic compounds.
Only a few organisms—the cyanobacteria and many FIGURE 2 Cycling of nitrogen in the biosphere. Gaseous nitrogen (N2)
makes up 80% of the earth’s atmosphere.
species of soil bacteria that live symbiotically on the
roots of some plants—are capable of converting (“fix-
ing”) atmospheric nitrogen (N2) into ammonia. Other heterotrophic organisms. In metabolic processes, and
bacteria (the nitrifying bacteria) oxidize ammonia to in all energy transformations, there is a loss of use-
nitrites and nitrates; yet others convert nitrate to N2. ful energy (free energy) and an inevitable increase in
The anammox bacteria convert ammonia and nitrite the amount of unusable energy (heat and entropy).
to N2. Thus, in addition to the global carbon and oxy- In contrast to the cycling of matter, therefore, energy
gen cycles, a nitrogen cycle operates in the biosphere, flows one way through the biosphere; organisms can-
turning over huge amounts of nitrogen (Fig. 2). The not regenerate useful energy from energy dissipated as
cycling of carbon, oxygen, and nitrogen, which ulti- heat and entropy. Carbon, oxygen, and nitrogen recycle
mately involves all species, depends on a proper balance continuously, but energy is constantly transformed into
between the activities of the producers (autotrophs) unusable forms such as heat.
and consumers (heterotrophs) in our biosphere. Metabolism, the sum of all the chemical trans-
These cycles of matter are driven by an enormous formations taking place in a cell or organism, occurs
flow of energy into and through the biosphere, begin- through a series of enzyme-catalyzed reactions that
ning with the capture of solar energy by photosyn- constitute metabolic pathways. Each of the consecu-
thetic organisms and use of this energy to generate tive steps in a metabolic pathway brings about a specific,
energy-rich carbohydrates and other organic nutrients; small chemical change, usually the removal, transfer, or
these nutrients are then used as energy sources by addition of a particular atom or functional group. The
precursor is converted into a product through a series
of metabolic intermediates called metabolites. The
FIGURE 1 Cycling of carbon dioxide and oxygen term intermediary metabolism is often applied to the
between the autotrophic (photosynthetic) and het-
combined activities of all the metabolic pathways that
erotrophic domains in the biosphere. The flow of
mass through this cycle is enormous; about 4 3 1011 interconvert precursors, metabolites, and products of
metric tons of carbon are turned over in the bio- low molecular weight (generally, Mr , 1,000).
sphere annually. Catabolism is the degradative phase of metabolism
in which organic nutrient molecules (carbohydrates,
O2
fats, and proteins) are converted into smaller, simpler end
gan
ic produ products (such as lactic acid, CO2, and NH3). Catabolic
Or ct
pathways release energy, some of which is conserved
s
Photosynthetic Heterotrophs
in the formation of ATP and reduced electron carriers
autotrophs
(NADH, NADPH, and FADH2); the rest is lost as heat.
In anabolism, also called biosynthesis, small, simple
CO2
precursors are built up into larger and more complex
H2O molecules, including lipids, polysaccharides, proteins,
Bioenergetics and Metabolism 503
and nucleic acids. Anabolic reactions require an input reaction sequences: when one sequence is active, the
of energy, generally in the form of the phosphoryl group other is suppressed. Such regulation could not occur
transfer potential of ATP and the reducing power of if anabolic and catabolic pathways were catalyzed by
NADH, NADPH, and FADH2 (Fig. 3). exactly the same set of enzymes, operating in one direc-
Some metabolic pathways are linear, and some are tion for anabolism, the opposite direction for catabo-
branched, yielding multiple useful end products from a lism: inhibition of an enzyme involved in catabolism
single precursor or converting several starting materials would also inhibit the reaction sequence in the anabolic
into a single product. In general, catabolic pathways are direction. Catabolic and anabolic pathways that connect
convergent and anabolic pathways divergent (Fig. 4). the same two end points (glucose S S pyruvate, and
Some pathways are cyclic: one starting component of pyruvate S S glucose, for example) may employ many
the pathway is regenerated in a series of reactions that of the same enzymes, but invariably at least one of the
converts another starting component into a product. steps is catalyzed by different enzymes in the catabolic
We shall see examples of each type of pathway in the and anabolic directions, and these enzymes are the sites
following chapters. of separate regulation. Moreover, for both anabolic and
Most cells have the enzymes to carry out both catabolic pathways to be essentially irreversible, the
the degradation and the synthesis of the important reactions unique to each direction must include at least
categories of biomolecules—fatty acids, for example. one that is thermodynamically very favorable—in other
The simultaneous synthesis and degradation of fatty words, a reaction for which the reverse reaction is very
acids would be wasteful, however, and this is prevented unfavorable. As a further contribution to the separate
by reciprocally regulating the anabolic and catabolic regulation of catabolic and anabolic reaction sequences,
paired catabolic and anabolic pathways commonly take
place in different cellular compartments: for example,
Cell Energy- fatty acid catabolism in mitochondria, fatty acid synthe-
macromolecules containing
nutrients sis in the cytosol. The concentrations of intermediates,
Proteins
Polysaccharides Carbohydrates enzymes, and regulators can be maintained at differ-
Lipids Fats
Nucleic acids Proteins ent levels in these different compartments. Because
metabolic pathways are subject to kinetic control by
substrate concentration, separate pools of anabolic and
catabolic intermediates also contribute to the control
of metabolic rates. Devices that separate anabolic and
ADP⫹ HPO2⫺
NAD⫹ catabolic processes will be of particular interest in our
NADP⫹
FAD
discussions of metabolism.
Metabolic pathways are regulated at several levels,
Anabolism ATP Catabolism from within the cell and from outside. The most immedi-
NADH ate regulation is by the availability of substrate; when the
NADPH
FADH2 intracellular concentration of an enzyme’s substrate is
near or below Km (as is commonly the case), the rate of
the reaction depends strongly on substrate concentra-
Chemical
energy tion (see Fig. 6–11). A second type of rapid control from
within is allosteric regulation (p. 226) by a metabolic
intermediate or coenzyme—an amino acid or ATP, for
Precursor Energy- example—that signals the cell’s internal metabolic state.
molecules depleted
Amino acids end products When the cell contains an amount of, say, aspartate suf-
Sugars CO2 ficient for its immediate needs or when the cellular level
Fatty acids H2O
Nitrogenous bases NH3
of ATP indicates that further fuel consumption is unnec-
essary at the moment, these signals allosterically inhibit
the activity of one or more enzymes in the relevant
FIGURE 3 Energy relationships between catabolic and anabolic pathways.
pathway. In multicellular organisms, the metabolic activ-
Catabolic pathways deliver chemical energy in the form of ATP, NADH,
NADPH, and FADH2. These energy carriers are used in anabolic path- ities of different tissues are regulated and integrated
ways to convert small precursor molecules into cellular macromolecules. by growth factors and hormones that act from outside
504 Bioenergetics and Metabolism
Rubber Carotenoid Steroid

pigments hormones
Phospholipids Isopentenyl- Bile

pyrophosphate Cholesterol
acids
Triacylglycerols Fatty acids
Mevalonate Vitamin K Cholesteryl

Phenyl- esters
Starch Alanine
alanine
Acetate
Glycogen Glucose Pyruvate (acetyl-CoA) Acetoacetyl-CoA Eicosanoids
Sucrose Serine Leucine

Isoleucine Fatty acids Triacylglycerols
(a) Converging catabolism

Citrate CDP-diacylglycerol Phospholipids
Oxaloacetate (b) Diverging anabolism
FIGURE 4 Three types of nonlinear metabolic pathways. (a)
Converging, catabolic, (b) diverging, anabolic, and (c) cyclic
pathways. In (c), one of the starting materials (oxaloacetate
CO2
in this case) is regenerated and reenters the pathway. Ace-
tate, a key metabolic intermediate, is the breakdown product
of a variety of fuels (a), serves as the precursor for an array
CO2 of products (b), and is consumed in the catabolic pathway
(c) Cyclic pathway known as the citric acid cycle (c).
the cell. In some cases, this regulation occurs virtually nucleotides from simpler precursors. In Chapter 23 we
instantaneously (sometimes in less than a millisecond) step back from our detailed look at the metabolic path-
through changes in the levels of intracellular messengers ways—as they occur in all organisms, from Escherichia
that modify the activity of existing enzyme molecules by coli to humans—and consider how they are regulated
allosteric mechanisms or by covalent modification such and integrated in mammals by hormonal mechanisms.
as phosphorylation. In other cases, the extracellular sig- As we undertake our study of intermediary metabo-
nal changes the cellular concentration of an enzyme by lism, a final word. Keep in mind that the myriad reac-
altering the rate of its synthesis or degradation, so the tions described in these pages take place in, and play
effect is seen only after minutes or hours. crucial roles in, living organisms. As you encounter
We begin Part II with a discussion of the basic ener- each reaction and each pathway, ask, What does this
getic principles that govern all metabolism (Chapter chemical transformation do for the organism? How does
13). We then consider the major catabolic pathways by this pathway interconnect with the other pathways
which cells obtain energy from the oxidation of various operating simultaneously in the same cell to produce
fuels (Chapters 14 through 19). Chapter 19 is the piv- the energy and products required for cell mainte-
otal point of our discussion of metabolism; it concerns nance and growth? How do the multilayered regulatory
chemiosmotic energy coupling, a universal mechanism mechanisms cooperate to balance metabolic and energy
in which a transmembrane electrochemical potential, inputs and outputs, achieving the dynamic steady state
produced either by substrate oxidation or by light of life? Studied with this perspective, metabolism pro-
absorption, drives the synthesis of ATP. vides fascinating and revealing insights into life, with
Chapters 20 through 22 describe the major ana- countless applications in medicine, agriculture, and
bolic pathways by which cells use the energy in ATP biotechnology.
to produce carbohydrates, lipids, amino acids, and
14
Glycolysis, Gluconeogenesis, and
the Pentose Phosphate Pathway
14.1 Glycolysis 544 nucleic acid synthesis and NADPH for reductive
biosynthetic processes (Fig. 14–1).
14.2 Feeder Pathways for Glycolysis 558 Organisms that do not have access to glucose from
14.3 Fates of Pyruvate under Anaerobic Conditions: other sources must make it. Photosynthetic organisms
Fermentation 563 make glucose by first reducing atmospheric CO2 to tri-
oses, then converting the trioses to glucose. Nonphoto-
14.4 Gluconeogenesis 568 synthetic cells make glucose from simpler three- and
14.5 Pentose Phosphate Pathway of Glucose Oxidation 575 four-carbon precursors by the process of gluconeogen-
esis, effectively reversing glycolysis in a pathway that
uses many of the glycolytic enzymes.
G
lucose occupies a central position in the metabo- In this chapter we describe the individual reactions
lism of plants, animals, and many microorganisms. of glycolysis, gluconeogenesis, and the pentose phos-
It is relatively rich in potential energy, and thus a phate pathway and the functional significance of each
good fuel; the complete oxidation of glucose to carbon pathway. We also describe the various metabolic fates
dioxide and water proceeds with a standard free-energy of the pyruvate produced by glycolysis. They include
change of 22,840 kJ/mol. By storing glucose as a high the fermentations that are used by many organisms in
molecular weight polymer such as starch or glycogen, a anaerobic niches to produce ATP and that are exploited
cell can stockpile large quantities of hexose units while industrially as sources of ethanol, lactic acid, and other
maintaining a relatively low cytosolic osmolarity. When commercially useful products. And we look at the pathways
energy demands increase, glucose can be released from that feed various sugars from mono-, di-, and polysac-
these intracellular storage polymers and used to pro- charides into the glycolytic pathway. The discussion of
duce ATP either aerobically or anaerobically. glucose metabolism continues in Chapter 15, where we
Glucose is not only an excellent fuel, it is also a
remarkably versatile precursor, capable of supplying a
Extracellular matrix
huge array of metabolic intermediates for biosynthetic and cell wall Glycogen,
reactions. A bacterium such as Escherichia coli can polysaccharides starch, sucrose
obtain from glucose the carbon skeletons for every
amino acid, nucleotide, coenzyme, fatty acid, or other synthesis of
metabolic intermediate it needs for growth. A compre- structural storage
polymers
hensive study of the metabolic fates of glucose would
encompass hundreds or thousands of transformations. Glucose
In animals and vascular plants, glucose has four major oxidation via
oxidation via
fates: it may be used in the synthesis of complex poly- pentose phosphate
glycolysis
pathway
saccharides destined for the extracellular space;
stored in cells (as a polysaccharide or as sucrose);
Ribose 5-phosphate Pyruvate
oxidized to a three-carbon compound (pyruvate) via
glycolysis to provide ATP and metabolic intermedi- FIGURE 14–1 Major pathways of glucose utilization. Although not the only
ates; or oxidized via the pentose phosphate (phospho- possible fates for glucose, these four pathways are the most significant in
gluconate) pathway to yield ribose 5-phosphate for terms of the amount of glucose that flows through them in most cells.
543
544 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
use the processes of carbohydrate synthesis and degra- carbon in most cells. The glycolytic breakdown of glucose
dation to illustrate the many mechanisms by which is the sole source of metabolic energy in some mamma-
organisms regulate metabolic pathways. The biosyn- lian tissues and cell types (erythrocytes, renal medulla,
thetic pathways from glucose to extracellular matrix brain, and sperm, for example). Some plant tissues that
and cell wall polysaccharides and storage polysaccha- are modified to store starch (such as potato tubers) and
rides are discussed in Chapter 20. some aquatic plants (watercress, for example) derive
most of their energy from glycolysis; many anaerobic
microorganisms are entirely dependent on glycolysis.
14.1 Glycolysis Fermentation is a general term for the anaerobic
In glycolysis (from the Greek glykys, “sweet” or degradation of glucose or other organic nutrients to
“sugar,” and lysis, “splitting”), a molecule of glucose is obtain energy, conserved as ATP. Because living organ-
degraded in a series of enzyme-catalyzed reactions to isms first arose in an atmosphere without oxygen, anaero-
yield two molecules of the three-carbon compound pyru- bic breakdown of glucose is probably the most ancient
vate. During the sequential reactions of glycolysis, some biological mechanism for obtaining energy from organic
of the free energy released from glucose is conserved in fuel molecules. And as genome sequencing of a wide vari-
the form of ATP and NADH. Glycolysis was the first ety of organisms has revealed, some archaea and some
metabolic pathway to be elucidated and is probably the parasitic microorganisms lack one or more of the enzymes
best understood. From Eduard Buchner’s discovery in of glycolysis but retain the core of the pathway; they pre-
1897 of fermentation in broken extracts of yeast cells sumably carry out variant forms of glycolysis. In the
until the elucidation of the whole pathway in yeast (by course of evolution, the chemistry of this reaction
Otto Warburg and Hans von Euler-Chelpin) and in sequence has been completely conserved; the glycolytic
muscle (by Gustav Embden and Otto Meyerhof) in the enzymes of vertebrates are closely similar, in amino acid
1930s, the reactions of glycolysis in extracts of yeast and sequence and three-dimensional structure, to their homo-
muscle were a major focus of biochemical research. The logs in yeast and spinach. Glycolysis differs among species
philosophical shift that accompanied these discoveries only in the details of its regulation and in the subsequent
was announced by Jacques Loeb in 1906: metabolic fate of the pyruvate formed. The thermody-
namic principles and the types of regulatory mechanisms
Through the discovery of Buchner, Biology was
that govern glycolysis are common to all pathways of cell
relieved of another fragment of mysticism. The
metabolism. The glycolytic pathway, of central impor-
splitting up of sugar into CO2 and alcohol is no more
tance in itself, can also serve as a model for many aspects
the effect of a “vital principle” than the splitting up
of the pathways discussed throughout this book.
of cane sugar by invertase. The history of this prob-
Before examining each step of the pathway in some
lem is instructive, as it warns us against considering
detail, we take a look at glycolysis as a whole.
problems as beyond our reach because they have
not yet found their solution.
The development of methods of enzyme purifica- An Overview: Glycolysis Has Two Phases
tion, the discovery and recognition of the importance of The breakdown of the six-carbon glucose into two mol-
coenzymes such as NAD, and the discovery of the piv- ecules of the three-carbon pyruvate occurs in 10 steps,
otal metabolic role of ATP and other phosphorylated the first 5 of which constitute the preparatory phase
compounds all came out of studies of glycolysis. The (Fig. 14–2a). In these reactions, glucose is first phos-
glycolytic enzymes of many species have long since phorylated at the hydroxyl group on C-6 (step 1). The
been purified and thoroughly studied. D-glucose 6-phosphate thus formed is converted to
Glycolysis is an almost universal central pathway of D-fructose 6-phosphate (step 2), which is again phos-
glucose catabolism, the pathway with the largest flux of phorylated, this time at C-1, to yield D-fructose
1,6-bisphosphate (step 3). For both
phosphorylations, ATP is the phospho-
ryl group donor. As all sugar derivatives
in glycolysis are the D isomers, we will
usually omit the D designation except
when emphasizing stereochemistry.
Fructose 1,6-bisphosphate is split to
yield two three-carbon molecules, dihy-
droxyacetone phosphate and glyceralde-
hyde 3-phosphate (step 4); this is the
“lysis” step that gives the pathway its
name. The dihydroxyacetone phosphate
Hans Von Euler-Chelpin, Gustav Embden, Otto Meyerhof, is isomerized to a second molecule of
1873–1964 1874–1933 1884–1951 glyceraldehyde 3-phosphate (step 5),
14.1 Glycolysis 545
6
(a) Preparatory phase HO CH2
5 O
Phosphorylation of glucose H H
and its conversion to Glucose H
4 1
glyceraldehyde 3-phosphate OH H
HO OH
1 3 2
ATP H OH
first priming reaction hexokinase 6
ADP P O CH2
5 O
Glucose 6-phosphate H H
H
4 1
OH H
HO OH
2 3 2
phosphohexose H OH
isomerase
6 1
P O CH2 O CH2 OH
Fructose 6-phosphate 5 2
H HO
H OH
ATP
3 4 3
second priming reaction phospho- OH H
fructokinase-1
ADP 6 1
P O CH2 O CH2 O P
Fructose 1,6-bisphosphate
5 2
H HO
cleavage of 6-carbon sugar H OH
phosphate to two 3-carbon 4 4 3
OH H
sugar phosphates aldolase
O
Glyceraldehyde 3-phosphate P O CH2 CH C
+ OH H
Dihydroxyacetone phosphate
5 P O CH2 C CH2 OH
triose phosphate
isomerase O
(b) Payof phase O

Oxidative conversion of (2) Glyceraldehyde (2) P O CH2 CH C
glyceraldehyde 3-phosphate 3-phosphate
OH H
to pyruvate and the coupled
formation of ATP and NADH 2Pi 6
2NAD⫹ glyceraldehyde
oxidation and 3-phosphate
O
phosphorylation dehydrogenase
2 NADH ⫹ 2H⫹ (2) P O CH2 CH C
(2) 1,3-Bisphosphoglycerate OH O P
first ATP-forming reaction 2ADP 7

(substrate-level phosphoglycerate O
kinase
phosphorylation) 2 ATP (2) P O CH2 CH C
OH O⫺
(2) 3-Phosphoglycerate
8 O
phosphoglycerate
mutase (2) CH2 CH C
(2) 2-Phosphoglycerate OH O O⫺
P
9 O
2H2O enolase
(2) CH2 C C
(2) Phosphoenolpyruvate O O⫺
second ATP-forming reaction 2ADP P

(substrate-level pyruvate
phosphorylation) kinase O
2 ATP
(2) CH3 C C
(2) Pyruvate O⫺
O
FIGURE 14–2 The two phases of glycolysis. For each molecule of glu- phase and four ATP are produced in the payoff phase, giving a net yield
cose that passes through the preparatory phase (a), two molecules of of two ATP per molecule of glucose converted to pyruvate. The num-
glyceraldehyde 3-phosphate are formed; both pass through the payoff bered reaction steps correspond to the numbered headings in the text
phase (b). Pyruvate is the end product of the second phase of glycolysis. discussion. Keep in mind that each phosphoryl group, represented here
For each glucose molecule, two ATP are consumed in the preparatory as P , has two negative charges (2PO232 ) .
ending the first phase of glycolysis. Note that two mole- lactate is also the product of glycolysis under anaerobic
cules of ATP are invested before the cleavage of glucose conditions in some microorganisms (Fig. 14–4).
into two three-carbon pieces; there will be a good return The third major route of pyruvate catabolism leads
on this investment. To summarize: in the preparatory to ethanol. In some plant tissues and in certain inver-
phase of glycolysis the energy of ATP is invested, raising tebrates, protists, and microorganisms such as brew-
the free-energy content of the intermediates, and the car- er’s or baker’s yeast, pyruvate is converted under
bon chains of all the metabolized hexoses are converted to hypoxic or anaerobic conditions to ethanol and CO2, a
a common product, glyceraldehyde 3-phosphate. process called ethanol (alcohol) fermentation
The energy gain comes in the payoff phase of gly- (Fig. 14–4).
colysis (Fig. 14–2b). Each molecule of glyceraldehyde The oxidation of pyruvate is an important catabolic
3-phosphate is oxidized and phosphorylated by inor- process, but pyruvate has anabolic fates as well. It can,
ganic phosphate (not by ATP) to form 1,3-bisphospho- for example, provide the carbon skeleton for the syn-
glycerate (step 6). Energy is then released as the two thesis of the amino acid alanine or for the synthesis of
molecules of 1,3-bisphosphoglycerate are converted to fatty acids. We return to these anabolic reactions of
two molecules of pyruvate (steps 7 through 10 ). Much pyruvate in later chapters.
of this energy is conserved by the coupled phosphoryla-
tion of four molecules of ADP to ATP. The net yield is ATP and NADH Formation Coupled to Glycolysis During gly-
two molecules of ATP per molecule of glucose used, colysis some of the energy of the glucose molecule is
because two molecules of ATP were invested in the conserved in ATP, while much remains in the product,
preparatory phase. Energy is also conserved in the pay- pyruvate. The overall equation for glycolysis is
off phase in the formation of two molecules of the elec-
Glucose 1 2NAD1 1 2ADP 1 2Pi 88n
tron carrier NADH per molecule of glucose.
2 pyruvate 1 2NADH 1 2H1 1 2ATP 1 2H2O (14–1)
In the sequential reactions of glycolysis, three
types of chemical transformations are particularly For each molecule of glucose degraded to pyruvate, two
noteworthy: (1) degradation of the carbon skeleton of molecules of ATP are generated from ADP and Pi, and
glucose to yield pyruvate; (2) phosphorylation of ADP two molecules of NADH are produced by the reduction
to ATP by compounds with high phosphoryl group of NAD1. The hydrogen acceptor in this reaction is
transfer potential, formed during glycolysis; and (3) NAD1 (see Fig. 13–24), bound to a Rossmann fold as
transfer of a hydride ion to NAD1, forming NADH. The shown in Figure 13–25. The reduction of NAD1 pro-
overall chemical logic of the pathway is described in ceeds by the enzymatic transfer of a hydride ion (:H2)
Figure 14–3. from the aldehyde group of glyceraldehyde 3-phosphate
to the nicotinamide ring of NAD1, yielding the reduced
Fates of Pyruvate With the exception of some interesting coenzyme NADH. The other hydrogen atom of the sub-
variations in the bacterial realm, the pyruvate formed strate molecule is released to the solution as H1.
by glycolysis is further metabolized via one of three We can now resolve the equation of glycolysis into
catabolic routes. In aerobic organisms or tissues, under two processes—the conversion of glucose to pyruvate,
aerobic conditions, glycolysis is only the first stage which is exergonic:
in the complete degradation of glucose (Fig. 14–4).
Glucose 1 2NAD1 88n 2 pyruvate 1 2NADH 1 2H1 (14–2)
Pyruvate is oxidized, with loss of its carboxyl group as
DG918 5 2146 kJ/mol
CO2, to yield the acetyl group of acetyl-coenzyme A; the
acetyl group is then oxidized completely to CO2 by the and the formation of ATP from ADP and Pi, which is
citric acid cycle (Chapter 16). The electrons from these endergonic:
oxidations are passed to O2 through a chain of carri-
2ADP 1 2Pi 88n 2ATP 1 2H2O (14–3)
ers in mitochondria, to form H2O. The energy from the
DG928 5 2(30.5 kJ/mol) 5 61.0 kJ/mol
electron-transfer reactions drives the synthesis of ATP
in mitochondria (Chapter 19). The sum of Equations 14–2 and 14–3 gives the overall
The second route for pyruvate is its reduction to lac- standard free-energy change of glycolysis, DG9s8:
tate via lactic acid fermentation. When vigorously
¢G¿s8 5 ¢G¿18 1 ¢G¿28 5 2146 kJ/mol 1 61.0 kJ/mol
contracting skeletal muscle must function under low-
5 285 kJ/mol
oxygen conditions (hypoxia), NADH cannot be reoxi-
dized to NAD1, but NAD1 is required as an electron Under standard conditions, and under the (nonstan-
acceptor for the further oxidation of pyruvate. Under dard) conditions that prevail in a cell, glycolysis is an
these conditions pyruvate is reduced to lactate, accepting essentially irreversible process, driven to completion by
electrons from NADH and thereby regenerating the a large net decrease in free energy.
NAD1 necessary for glycolysis to continue. Certain tissues
and cell types (retina and erythrocytes, for example) con- Energy Remaining in Pyruvate Glycolysis releases only a
vert glucose to lactate even under aerobic conditions, and small fraction of the total available energy of the glucose
14.1 Glycolysis 547
1 2 3 4 5 6 Phosphorylation molecule; the two molecules of pyruvate formed by

O OH OH OH OH of glucose glycolysis still contain most of the chemical potential
Glucose ensures that energy of glucose, energy that can be extracted by
pathway
OH intermediates oxidative reactions in the citric acid cycle (Chapter 16)
1 Phosphorylation occurs on C-6, remain in the cell. and oxidative phosphorylation (Chapter 19).
ATP
as C-1 is a carbonyl group and
cannot be phosphorylated. ADP Importance of Phosphorylated Intermediates Each of the
nine glycolytic intermediates between glucose and
O OH OH OH O P Glucose pyruvate is phosphorylated (Fig. 14–2). The phosphoryl
6-phosphate
groups seem to have three functions.
OH
Isomerization moves the carbonyl to C-2, a
1. Because the plasma membrane generally lacks
2
prerequisite for 3 and 4. transporters for phosphorylated sugars, the
phosphorylated glycolytic intermediates cannot
HO O OH OH O P Fructose leave the cell. After the initial phosphorylation, no
6-phosphate
further energy is necessary to retain
ATP OH phosphorylated intermediates in the cell, despite
ADP 3 C-1, now a hydroxyl group, can be the large difference in their intracellular and
phosphorylated. This ensures that both
products of C–C bond cleavage are
extracellular concentrations.
phosphorylated, and thus interconvertible.
2. Phosphoryl groups are essential components in the
P O O OH OH O P Fructose enzymatic conservation of metabolic energy. Energy
1,6-bisphosphate released in the breakage of phosphoanhydride bonds
OH (such as those in ATP) is partially conserved in the
4 The carbonyl group at C-2
facilitates C–C bond cleavage at formation of phosphate esters such as glucose
Dihydroxyacetone
phosphate
the right location to yield two 6-phosphate. High-energy phosphate compounds
3-carbon products by the reverse formed in glycolysis (1,3-bisphosphoglycerate and
P O O OH of an aldol condensation.
phosphoenolpyruvate) donate phosphoryl groups to
Glyceraldehyde ADP to form ATP.
3-phosphate
O OH O P 3. Binding energy resulting from the binding of
5 Interconversion
of the two
phosphate groups to the active sites of enzymes
products of 4 6 Oxidative phosphorylation of lowers the activation energy and increases the
funnels both glyceraldehyde 3-phosphate, specificity of the enzymatic reactions (Chapter 6).
products into a with one NADH produced, is
single pathway.
NAD⫹, Pi
a prerequisite for ATP
The phosphate groups of ADP, ATP, and the
NADH production in 7. glycolytic intermediates form complexes with
Mg21, and the substrate binding sites of many
O OH O P glycolytic enzymes are specific for these Mg21
1,3-Bisphosphoglycerate complexes. Most glycolytic enzymes require Mg21
P O
for activity.
7 ATP production
ADP
ATP O OH O P
3-Phosphoglycerate
⫺
O
8 The remaining phosphoryl
group moves from C-2 to
C-3, setting up the final
steps of the pathway.
O O P
2-Phosphoglycerate
⫺
O
FIGURE 14–3 The chemical logic of the glycolytic pathway. In this sim-
OH plified version of the pathway, each molecule is shown in a linear form,
9 Dehydration activates
the phosphoryl for with carbon and hydrogen atoms not depicted, in order to highlight chem-
transfer to ADP in 1. ical transformations. Remember that glucose and fructose are present
O O P
Phosphoenolpyruvate mostly in their cyclized forms in solution, although they are transiently
⫺
O present in linear form at the active sites of some of the enzymes in this
ADP
ATP production pathway.
ATP The preparatory phase, steps 1 to 5, converts the six-carbon glu-
O O cose into two three-carbon units, each of them phosphorylated. Oxida-
Pyruvate tion of the three-carbon units is initiated in the payoff phase. To produce
⫺
O pyruvate, the chemical steps must occur in the order shown.
Glucose responsible for the stimulation. Harden and Young soon

discovered that glucose added to their yeast extract was
glycolysis converted to a hexose bisphosphate (the “Harden-
(10 successive
reactions)
Young ester,” eventually identified as fructose 1,6-bispho-
hypoxic or hypoxic or sphate). This was the beginning of a long series of
anaerobic anaerobic
conditions 2 Pyruvate conditions
investigations on the role of organic esters and anhy-
drides of phosphate in biochemistry, which has led to
aerobic our current understanding of the central role of phos-
2 Ethanol ⫹ 2CO2
conditions
2 Lactate phoryl group transfer in biology.
2CO2
Fermentation to Fermentation to lactate 1 Phosphorylation of Glucose In the first step of glycoly-
ethanol in yeast 2 Acetyl-CoA in vigorously contracting
muscle, in erythrocytes, sis, glucose is activated for subsequent reactions by its
in some other cells, and phosphorylation at C-6 to yield glucose 6-phosphate,
citric
acid in some microorganisms with ATP as the phosphoryl donor:
cycle
6
CH2 O OPO32⫺
4CO2 ⫹ 4H2O CH2 O OH
5
O ATP ADP O
H H H H
Animal, plant, and H Mg2⫹ H
4 1
many microbial OH H hexokinase OH H
cells under aerobic HO OH HO OH
conditions 3 2
H OH H OH
FIGURE 14–4 Three possible catabolic fates of the pyruvate formed in Glucose Glucose 6-phosphate
glycolysis. Pyruvate also serves as a precursor in many anabolic reac-
tions, not shown here.
DG⬘⬚ ⫽ ⫺16.7 kJ/mol
This reaction, which is irreversible under intracellular con-

The Preparatory Phase of Glycolysis Requires ATP ditions, is catalyzed by hexokinase. Recall that kinases
In the preparatory phase of glycolysis, two molecules of are enzymes that catalyze the transfer of the terminal
ATP are invested and the hexose chain is cleaved into two phosphoryl group from ATP to an acceptor nucleophile
triose phosphates. The realization that phosphorylated (see Fig. 13–20). Kinases are a subclass of transferases
hexoses were intermediates in glycolysis came slowly (see Table 6–3). The acceptor in the case of hexokinase is
and serendipitously. In 1906, Arthur Harden and William a hexose, normally D-glucose, although hexokinase also
Young tested their hypothesis that inhibitors catalyzes the phosphorylation of other common hexoses,
of proteolytic enzymes would stabilize the glucose- such as D-fructose and D-mannose, in some tissues.
fermenting enzymes in yeast extract. They added blood Hexokinase, like many other kinases, requires Mg21
serum (known to contain inhibitors of proteolytic for its activity, because the true substrate of the enzyme
enzymes) to yeast extracts and observed the predicted is not ATP42 but the MgATP22 complex (see Fig. 13–12).
stimulation of glucose metabolism. However, in a con- Mg21 shields the negative charges of the phosphoryl
trol experiment intended to show that boiling the serum groups in ATP, making the terminal phosphorus atom an
destroyed the stimulatory activity, they discovered that easier target for nucleophilic attack by an —OH of glu-
boiled serum was just as effective at stimulating glycoly- cose. Hexokinase undergoes a profound change in shape,
sis! Careful examination and testing of the contents of an induced fit, when it binds glucose; two domains of the
the boiled serum revealed that inorganic phosphate was protein move about 8 Å closer to each other when ATP
binds (see Fig. 6–25). This movement brings bound ATP
closer to a molecule of glucose also bound to the enzyme
and blocks the access of water (from the solvent), which
might otherwise enter the active site and attack (hydro-
lyze) the phosphoanhydride bonds of ATP. Like the other
nine enzymes of glycolysis, hexokinase is a soluble, cyto-
solic protein.
Hexokinase is present in nearly all organisms. The
human genome encodes four different hexokinases (I to
IV), all of which catalyze the same reaction. Two or
more enzymes that catalyze the same reaction but are
encoded by different genes are called isozymes (see
Arthur Harden, William Young, Box 15–2). One of the isozymes present in hepatocytes,
1865–1940 1878–1942 hexokinase IV (also called glucokinase), differs from
602 Principles of Metabolic Regulation
Hexokinase Isozymes of Muscle and Liver Are myocytes from the blood (where the glucose concen-
tration is 4 to 5 mM) produces an intracellular glucose
Affected Differently by Their Product, Glucose
concentration high enough to saturate hexokinase II,
6-Phosphate the enzyme normally acts at or near its maximal rate.
Hexokinase, which catalyzes the entry of glucose into Muscle hexokinase I and hexokinase II are allosteri-
the glycolytic pathway, is a regulatory enzyme. Humans cally inhibited by their product, glucose 6-phosphate,
have four isozymes (designated I to IV), encoded by so whenever the cellular concentration of glucose
four different genes. Isozymes are different proteins 6-phosphate rises above its normal level, these iso-
that catalyze the same reaction (Box 15–2). The zymes are temporarily and reversibly inhibited, bringing
predominant hexokinase isozyme of myocytes (hexo- the rate of glucose 6-phosphate formation into balance
kinase II) has a high affinity for glucose—it is half- with the rate of its utilization and reestablishing the
saturated at about 0.1 mM. Because glucose entering steady state.
BOX 15–2 Isozymes: Different Proteins That Catalyze the Same Reaction
The four forms of hexokinase found in mammalian there is more LDH2 than LDH1. After 12 hours the
tissues are but one example of a common biological amounts of LDH1 and LDH2 are very similar, and after
situation: the same reaction catalyzed by two or more 24 hours there is more LDH1 than LDH2. This switch in
different molecular forms of an enzyme. These multi- the [LDH1]/[LDH2] ratio, combined with increased con-
ple forms, called isozymes or isoenzymes, may occur centrations in the blood of another heart enzyme,
in the same species, in the same tissue, even in the creatine kinase, is very strong evidence of a recent
same cell. The different forms (isoforms) of the myocardial infarction. ■
enzyme generally differ in kinetic or regulatory prop- The different LDH isozymes have significantly
erties, in the cofactor they use (NADH or NADPH for different values of Vmax and Km, particularly for pyru-
dehydrogenase isozymes, for example), or in their vate. The properties of LDH4 favor rapid reduction of
subcellular distribution (soluble or membrane- very low concentrations of pyruvate to lactate in skel-
bound). Isozymes may have similar, but not identical, etal muscle, whereas those of isozyme LDH1 favor
amino acid sequences, and in many cases they clearly rapid oxidation of lactate to pyruvate in the heart.
share a common evolutionary origin. In general, the distribution of different isozymes
One of the first enzymes found to have isozymes of a given enzyme reflects at least four factors:
was lactate dehydrogenase (LDH; p. 563), which in
1. Different metabolic patterns in different
vertebrate tissues exists as at least five different iso-
organs. For glycogen phosphorylase, the isozymes
zymes separable by electrophoresis. All LDH isozymes
in skeletal muscle and liver have different
contain four polypeptide chains (each of Mr 33,500),
regulatory properties, reflecting the different roles
each type containing a different ratio of two kinds of
of glycogen breakdown in these two tissues.
polypeptides. The M (for muscle) chain and the H
(for heart) chain are encoded by two different genes. 2. Different locations and metabolic roles for
In skeletal muscle the predominant isozyme con- isozymes in the same cell. The isocitrate
tains four M chains, and in heart the predominant iso- dehydrogenase isozymes of the cytosol and the
zyme contains four H chains. Other tissues have some mitochondrion are an example (Chapter 16).
combination of the five possible types of LDH isozymes:
3. Different stages of development in embryonic
Type Composition Location or fetal tissues and in adult tissues. For
example, the fetal liver has a characteristic
LDH1 HHHH Heart and erythrocyte isozyme distribution of LDH, which changes as
LDH2 HHHM Heart and erythrocyte
the organ develops into its adult form. Some
LDH3 HHMM Brain and kidney
LDH4
enzymes of glucose catabolism in malignant
HMMM Skeletal muscle and liver
LDH5 MMMM Skeletal muscle and liver (cancer) cells occur as their fetal, not adult,
isozymes.
The differences in the isozyme content of tis- 4. Different responses of isozymes to allosteric
sues can be used to assess the timing and extent modulators. This difference is useful in fine-
of heart damage due to myocardial infarction (heart tuning metabolic rates. Hexokinase IV
attack). Damage to heart tissue results in the release (glucokinase) of liver and the hexokinase
of heart LDH into the blood. Shortly after a heart isozymes of other tissues differ in their
attack, the blood level of total LDH increases, and sensitivity to inhibition by glucose 6-phosphate.
15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 603
The different hexokinase isozymes of liver and

muscle reflect the different roles of these organs in car- 1.0
bohydrate metabolism: muscle consumes glucose, using Hexokinase I
Relative enzyme activity

it for energy production, whereas liver maintains blood
glucose homeostasis by consuming or producing glu-
cose, depending on the prevailing blood glucose con-
Hexokinase IV
centration. The predominant hexokinase isozyme of (glucokinase)
liver is hexokinase IV (glucokinase), which differs in
three important respects from hexokinases I–III of
muscle. First, the glucose concentration at which hexo-
kinase IV is half-saturated (about 10 mM) is higher than
the usual concentration of glucose in the blood. Because 0 5 10 15 20
an efficient glucose transporter in hepatocytes (GLUT2) Glucose concentration (mM)
rapidly equilibrates the glucose concentrations in cyto-
FIGURE 15–14 Comparison of the kinetic properties of hexokinase IV
sol and blood (see Fig. 11–31 for the kinetics of the (glucokinase) and hexokinase I. Note the sigmoidicity for hexokinase IV
same transporter, GLUT1, in erythrocytes), the high Km and the much lower Km for hexokinase I. When blood glucose rises
of hexokinase IV allows its direct regulation by the level above 5 mM, hexokinase IV activity increases, but hexokinase I is already
of blood glucose (Fig. 15–14). When blood glucose is operating near Vmax and cannot respond to an increase in glucose con-
high, as it is after a meal rich in carbohydrates, excess centration. Hexokinases I, II, and III have similar kinetic properties.
glucose is transported into hepatocytes, where hexoki-
nase IV converts it to glucose 6-phosphate. Because below 5 mM, fructose 6-phosphate triggers the inhibition
hexokinase IV is not saturated at 10 mM glucose, its of hexokinase IV by the regulatory protein, so the liver
activity continues to increase as the glucose concentra- does not compete with other organs for the scarce glu-
tion rises to 10 mM or more. Under conditions of low cose. The mechanism of inhibition by the regulatory
blood glucose, the glucose concentration in a hepato- protein is interesting: the protein anchors hexokinase IV
cyte is low relative to the Km of hexokinase IV, and the inside the nucleus, where it is segregated from the other
glucose generated by gluconeogenesis leaves the cell enzymes of glycolysis in the cytosol (Fig. 15–15). When
before being trapped by phosphorylation. the glucose concentration in the cytosol rises, it equili-
Second, hexokinase IV is not inhibited by glucose brates with glucose in the nucleus by transport through
6-phosphate, and it can therefore continue to operate the nuclear pores. Glucose causes dissociation of the
when the accumulation of glucose 6-phosphate com- regulatory protein, and hexokinase IV enters the cytosol
pletely inhibits hexokinases I–III. Finally, hexokinase IV and begins to phosphorylate glucose.
is subject to inhibition by the reversible binding of a
regulatory protein specific to liver (Fig. 15–15). The
binding is much tighter in the presence of the allosteric
Hexokinase IV (Glucokinase) and Glucose
effector fructose 6-phosphate. Glucose competes with 6-Phosphatase Are Transcriptionally Regulated
fructose 6-phosphate for binding and causes dissociation Hexokinase IV is also regulated at the level of protein
of the regulatory protein from the hexokinase, relieving synthesis. Circumstances that call for greater energy
the inhibition. Immediately after a carbohydrate-rich production (low [ATP], high [AMP], vigorous muscle con-
meal, when blood glucose is high, glucose enters the traction) or for greater glucose consumption (high blood
hepatocyte via GLUT2 and activates hexokinase IV by glucose, for example) cause increased transcription of
this mechanism. During a fast, when blood glucose drops the hexokinase IV gene. Glucose 6-phosphatase, the
Capillary
Cytosol Nucleus
GLUT2
Glucose
Glucose
FIGURE 15–15 Regulation of hexokinase IV
Plasma Hexokinase IV Hexokinase IV (glucokinase) by sequestration in the nucleus.
membrane The protein inhibitor of hexokinase IV is a nuclear
binding protein that draws hexokinase IV into
Regulatory
Glucose 6-phosphate protein the nucleus when the fructose 6-phosphate
concentration in liver is high and releases it to
the cytosol when the glucose concentration is
Fructose 6-phosphate
high.
gluconeogenic enzyme that bypasses the hexokinase step Allosteric

of glycolysis, is transcriptionally regulated by factors that regulators
(ATP)
call for increased production of glucose (low blood glu-
cose, glucagon signaling). The transcriptional regulation
of these two enzymes (along with other enzymes of Subunit 1
glycolysis and gluconeogenesis) is described below. ADP
Subunit 2
Phosphofructokinase-1 and Fructose
1,6-Bisphosphatase Are Reciprocally Regulated
As we have noted, glucose 6-phosphate can flow either Fructose
into glycolysis or through any of several other pathways, 1,6-bis-
including glycogen synthesis and the pentose phos- phosphate Subunit 3
phate pathway. The metabolically irreversible reaction ADP
catalyzed by PFK-1 is the step that commits glucose to
glycolysis. In addition to its substrate-binding sites, this
complex enzyme has several regulatory sites at which
allosteric activators or inhibitors bind. Subunit 4
ATP is not only a substrate for PFK-1 but also an
end product of the glycolytic pathway. When high Allosteric
regulators
cellular [ATP] signals that ATP is being produced faster (ATP)
than it is being consumed, ATP inhibits PFK-1 by
(a)
binding to an allosteric site and lowering the affinity of
the enzyme for its substrate fructose 6-phosphate
(Fig. 15–16). ADP and AMP, which increase in con- Low [ATP]
centration as consumption of ATP outpaces produc-
PFK-1 activity
tion, act allosterically to relieve this inhibition by ATP.

These effects combine to produce higher enzyme activ-
ity when ADP or AMP accumulates and lower activity High [ATP]
when ATP accumulates.
Citrate (the ionized form of citric acid), a key inter-
mediate in the aerobic oxidation of pyruvate, fatty acids,
[Fructose 6-phosphate]
and amino acids, is also an allosteric regulator of PFK-1;
high citrate concentration increases the inhibitory effect (b)
of ATP, further reducing the flow of glucose through
glycolysis. In this case, as in several others encountered ATP AMP, ADP
later, citrate serves as an intracellular signal that the cell
is meeting its current needs for energy-yielding metabo-
Fructose 6- ⫹ ATP Fructose 1,6- ⫹ ADP
lism by the oxidation of fats and proteins. phosphate bisphosphate
The corresponding step in gluconeogenesis is the
conversion of fructose 1,6-bisphosphate to fructose
citrate fructose 2,6-
6-phosphate (Fig. 15–17). The enzyme that catalyzes bisphosphate
this reaction, FBPase-1, is strongly inhibited (allosteri- (c)
cally) by AMP; when the cell’s supply of ATP is low
(corresponding to high [AMP]), the ATP-requiring syn- FIGURE 15–16 Phosphofructokinase-1 (PFK-1) and its regulation.
thesis of glucose slows. (a) Surface contour image of E. coli PFK-1, showing portions of its four
Thus these opposing steps in the glycolytic and identical subunits (PDB ID 1PFK). Each subunit has its own catalytic site,
gluconeogenic pathways—those catalyzed by PFK-1 where the products ADP and fructose 1,6-bisphosphate (red and yellow
and FBPase-1—are regulated in a coordinated and stick structures, respectively) are almost in contact, and its own binding
sites for the allosteric regulator ATP, buried in the protein in the positions
reciprocal manner. In general, when sufficient concen-
indicated. (b) Allosteric regulation of muscle PFK-1 by ATP, shown by a
trations of acetyl-CoA or citrate (the product of acetyl-
substrate-activity curve. At low [ATP], the K0.5 for fructose 6-phosphate
CoA condensation with oxaloacetate) are present, or
is relatively low, enabling the enzyme to function at a high rate at rela-
when a high proportion of the cell’s adenylate is in the
tively low [fructose 6-phosphate]. (Recall from Chapter 6 that K0.5 is the
form of ATP, gluconeogenesis is favored. When the
Km term for regulatory enzymes.) When [ATP] is high, K0.5 for fructose
level of AMP increases, it promotes glycolysis by stimu- 6-phosphate is greatly increased, as indicated by the sigmoid relation-
lating PFK-1 (and, as we shall see in Section 15.5, pro- ship between substrate concentration and enzyme activity. (c) Summary
motes glycogen degradation by activating glycogen of the regulators affecting PFK-1 activity.
phosphorylase).
Gluconeogenesis needs. One source of glucose is glycogen stored in the

liver; another source is gluconeogenesis, using pyru-
vate, lactate, glycerol, or certain amino acids as starting
ATP Pi
material. When blood glucose is high, insulin signals the
ATP
liver to use glucose as a fuel and as a precursor for the
ADP synthesis and storage of glycogen and triacylglycerol.
PFK-1 FBPase-1
AMP The rapid hormonal regulation of glycolysis and
citrate gluconeogenesis is mediated by fructose 2,6-bisphos-
ADP Fructose 1,6-bisphosphate H2O phate, an allosteric effector for the enzymes PFK-1 and
FBPase-1:
Glycolysis O O
B B
FIGURE 15–17 Regulation of fructose 1,6-bisphosphatase (FBPase-1)

O OP O OOCH2 O OP O O
A A
and phosphofructokinase-1 (PFK-1). The important role of fructose O O O
2,6-bisphosphate in the regulation of this substrate cycle is detailed in H HO
subsequent figures. H CH2OH
Fructose 2,6-Bisphosphate Is a Potent Allosteric OH H

Regulator of PFK-1 and FBPase-1
The special role of the liver in maintaining a constant When fructose 2,6-bisphosphate binds to its allosteric
blood glucose level requires additional regulatory mech- site on PFK-1, it increases the enzyme’s affinity for its
anisms to coordinate glucose production and consump- substrate fructose 6-phosphate and reduces its affinity
tion. When the blood glucose level decreases, the hor- for the allosteric inhibitors ATP and citrate (Fig. 15–18).
mone glucagon signals the liver to produce and release At the physiological concentrations of its substrates, ATP
more glucose and to stop consuming it for its own and fructose 6-phosphate, and of its other positive and
100 100
FBPase-1 activity (% of Vmax)
PFK-1 activity (% of Vmax)
80 ⫹F26BP
80
⫺F26BP
60 60
40 40
20 ⫺F26BP 20 ⫹F26BP
0 0
0 0.05 0.1 0.2 0.4 0.7 1.0 2.0 4.0 0 50 100
[Fructose 6-phosphate] (mM) [Fructose 1,6-bisphosphate] (␮M)
(a) (b)
Gluconeogenesis
ATP Fructose 6-phosphate Pi
PFK-1 F26BP FBPase-1
ADP Fructose 1,6-bisphosphate H2O
Glycolysis
(c)
FIGURE 15–18 Role of fructose 2,6-bisphosphate in regulation of gly- F26BP activates PFK-1 by increasing its apparent affinity for fructose
colysis and gluconeogenesis. Fructose 2,6-bisphosphate (F26BP) has 6-phosphate (see Fig. 15–16b). (b) FBPase-1 activity is inhibited by as
opposite effects on the enzymatic activities of phosphofructokinase-1 little as 1 M F26BP and is strongly inhibited by 25 M. In the absence of
(PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a this inhibitor (blue curve) the K0.5 for fructose 1,6-bisphosphate is 5 M,
gluconeogenic enzyme). (a) PFK-1 activity in the absence of F26BP (blue but in the presence of 25 M F26BP (red curve) the K0.5 is .70 M.
curve) is half-maximal when the concentration of fructose 6-phosphate is Fructose 2,6-bisphosphate also makes FBPase-1 more sensitive to inhi-
2 mM (that is, K0.5 5 2 mM). When 0.13 M F26BP is present (red bition by another allosteric regulator, AMP. (c) Summary of regulation
curve), the K0.5 for fructose 6-phosphate is only 0.08 mM. Thus by F26BP.
PFK-2
[F26BP] (active)
OH
Stimulates glycolysis, FBPase-2
inhibits gluconeogenesis (inactive)
Fructose 6-phosphate Pi ATP

ATP Pi
phospho-
cAMP-dependent glucagon
PFK-2 FBPase-2 insulin protein
protein kinase ( [cAMP])
phosphatase
ADP ADP
Fructose 2,6-bisphosphate H2O
(a) PFK-2 O
[F26BP] (inactive)
O P O
Inhibits glycolysis, FBPase-2
stimulates gluconeogenesis (active) O
(b)
FIGURE 15–19 Regulation of fructose 2,6-bisphosphate level. (a) The nase-2 (PFK-2) and its breakdown by fructose 2,6-bisphosphatase
cellular concentration of the regulator fructose 2,6-bisphosphate (FBPase-2). (b) Both enzyme activities are part of the same polypeptide
(F26BP) is determined by the rates of its synthesis by phosphofructoki- chain, and they are reciprocally regulated by insulin and glucagon.
negative effectors (ATP, AMP, citrate), PFK-1 is virtually Xylulose 5-Phosphate Is a Key Regulator
inactive in the absence of fructose 2,6-bisphosphate.
Fructose 2,6-bisphosphate has the opposite effect on
of Carbohydrate and Fat Metabolism
FBPase-1: it reduces its affinity for its substrate (Fig. Another regulatory mechanism also acts by controlling
15–18b), thereby slowing gluconeogenesis. the level of fructose 2,6-bisphosphate. In the mammalian
The cellular concentration of the allosteric regula- liver, xylulose 5-phosphate (p. 577), a product of the
tor fructose 2,6-bisphosphate is set by the relative rates pentose phosphate pathway (hexose monophosphate
of its formation and breakdown (Fig. 15–19a). It is pathway), mediates the increase in glycolysis that fol-
formed by phosphorylation of fructose 6-phosphate, lows ingestion of a high-carbohydrate meal. The xylulose
catalyzed by phosphofructokinase-2 (PFK-2), and is 5-phosphate concentration rises as glucose entering the
broken down by fructose 2,6-bisphosphatase liver is converted to glucose 6-phosphate and enters both
(FBPase-2). (Note that these enzymes are distinct the glycolytic and pentose phosphate pathways. Xylulose
from PFK-1 and FBPase-1, which catalyze the formation 5-phosphate activates phosphoprotein phosphatase 2A
and breakdown, respectively, of fructose 1,6-bisphos- (PP2A; Fig. 15–20), which dephosphorylates the bifunc-
phate.) PFK-2 and FBPase-2 are two separate enzy- tional PFK-2/FBPase-2 enzyme (Fig. 15–19). Dephos-
matic activities of a single, bifunctional protein. The phorylation activates PFK-2 and inhibits FBPase-2, and
balance of these two activities in the liver, which deter- the resulting rise in fructose 2,6-bisphosphate concen-
mines the cellular level of fructose 2,6-bisphosphate, is tration stimulates glycolysis and inhibits gluconeogen-
regulated by glucagon and insulin (Fig. 15–19b). esis. The increased glycolysis boosts the production of
As we saw in Chapter 12 (p. 446), glucagon stimu- acetyl-CoA, while the increased flow of hexose through
lates the adenylyl cyclase of liver to synthesize the pentose phosphate pathway generates NADPH.
39,59-cyclic AMP (cAMP) from ATP. Cyclic AMP then Acetyl-CoA and NADPH are the starting materials for
activates cAMP-dependent protein kinase, which trans- fatty acid synthesis, which has long been known to
fers a phosphoryl group from ATP to the bifunctional increase dramatically in response to intake of a high-
protein PFK-2/FBPase-2. Phosphorylation of this protein carbohydrate meal. Xylulose 5-phosphate also increases
enhances its FBPase-2 activity and inhibits its PFK-2 the synthesis of all the enzymes required for fatty acid
activity. Glucagon thereby lowers the cellular level of synthesis, meeting the prediction from metabolic con-
fructose 2,6-bisphosphate, inhibiting glycolysis and trol analysis. We return to this effect in our discussion
stimulating gluconeogenesis. The resulting production of the integration of carbohydrate and lipid metabolism
of more glucose enables the liver to replenish blood in Chapter 23.
glucose in response to glucagon. Insulin has the oppo-
site effect, stimulating the activity of a phosphoprotein
phosphatase that catalyzes removal of the phosphoryl
The Glycolytic Enzyme Pyruvate Kinase
group from the bifunctional protein PFK-2/FBPase-2, Is Allosterically Inhibited by ATP
activating its PFK-2 activity, increasing the level of At least three isozymes of pyruvate kinase are found in
fructose 2,6-bisphosphate, stimulating glycolysis, and vertebrates, differing in their tissue distribution and
inhibiting gluconeogenesis. their response to modulators. High concentrations of
(a)
Regulatory Catalytic subunit Scafold/A subunit
Catalytic Inhibitor subunit
subunit
2 Mn2+
Regulatory Regulatory
subunit 1 subunit 2
Scafold/
A subunit
Substrate- Substrate-
(a) recognition recognition
surface 1 surface 2
FIGURE 15–20 Structure and action of phosphoprotein phosphatase
2A (PP2A). (a) The catalytic subunit has two Mn21 ions in its active
site, positioned close to the substrate-recognition surface formed by
the interface between the catalytic subunit and the regulatory subunit
Holoenzyme 1 Holoenzyme 2
(PDB ID 2NPP). Microcystin-LR, shown here in red, is a specific inhib-
(b)
itor of PP2A. The catalytic and regulatory subunits rest in a scaffold
(the A subunit) that positions them relative to each other and shapes
the substrate-recognition site. (b) PP2A recognizes several target ATP, acetyl-CoA, and long-chain fatty acids (signs of
proteins, its specificity provided by the regulatory subunit. Each of abundant energy supply) allosterically inhibit all iso-
several regulatory subunits fits the scaffold containing the catalytic zymes of pyruvate kinase (Fig. 15–21). The liver iso-
subunit, and each regulatory subunit creates its unique substrate- zyme (L form), but not the muscle isozyme (M form), is
binding site. subject to further regulation by phosphorylation. When
low blood glucose causes glucagon release, cAMP-
dependent protein kinase phosphorylates the L isozyme
of pyruvate kinase, inactivating it. This slows the use of
Liver only All glycolytic tissues, including liver
glucagon
F16BP
ADP ATP 6 steps
PKA
PEP
P ADP
ATP,
acetyl-CoA,
long-chain fatty acids
Pyruvate Pyruvate ATP
kinase L kinase
Pyruvate
(inactive) PP L/M
H2O Pi transamination
Alanine
FIGURE 15–21 Regulation of pyruvate kinase. The enzyme is allosteri- cAMP-dependent protein kinase (PKA; see Fig. 15–37), which phosphor-
cally inhibited by ATP, acetyl-CoA, and long-chain fatty acids (all signs ylates the pyruvate kinase L isozyme, inactivating it. When the glucagon
of an abundant energy supply), and the accumulation of fructose level drops, a protein phosphatase (PP) dephosphorylates pyruvate
1,6-bisphosphate triggers its activation. Accumulation of alanine, which kinase, activating it. This mechanism prevents the liver from consuming
can be synthesized from pyruvate in one step, allosterically inhibits glucose by glycolysis when blood glucose is low; instead, the liver
pyruvate kinase, slowing the production of pyruvate by glycolysis. The exports glucose. The muscle isozyme (M form) is not affected by this
liver isozyme (L form) is also regulated hormonally. Glucagon activates phosphorylation mechanism.
glucose as a fuel in liver, sparing it for export to the accumulates. The increased concentration of acetyl-
brain and other organs. In muscle, the effect of increased CoA inhibits the pyruvate dehydrogenase complex,
[cAMP] is quite different. In response to epinephrine, slowing the formation of acetyl-CoA from pyruvate, and
cAMP activates glycogen breakdown and glycolysis and stimulates gluconeogenesis by activating pyruvate car-
provides the fuel needed for the fight-or-flight response. boxylase, allowing conversion of excess pyruvate to
oxaloacetate (and, eventually, glucose).
The Gluconeogenic Conversion of Pyruvate Oxaloacetate formed in this way is converted to
phosphoenolpyruvate (PEP) in the reaction catalyzed by
to Phosphoenolpyruvate Is under Multiple Types PEP carboxykinase (Fig. 15–13). In mammals, the regu-
of Regulation lation of this key enzyme occurs primarily at the level of
In the pathway leading from pyruvate to glucose, the its synthesis and breakdown, in response to dietary and
first control point determines the fate of pyruvate in the hormonal signals. Fasting or high glucagon levels act
mitochondrion: its conversion either to acetyl-CoA (by through cAMP to increase the rate of transcription and to
the pyruvate dehydrogenase complex) to fuel the citric stabilize the mRNA. Insulin, or high blood glucose, has
acid cycle (Chapter 16) or to oxaloacetate (by pyruvate the opposite effects. We discuss this transcriptional regu-
carboxylase) to start the process of gluconeogenesis lation in more detail below. Generally triggered by a sig-
(Fig. 15–22). When fatty acids are readily available as nal from outside the cell (diet, hormones), these changes
fuels, their breakdown in liver mitochondria yields take place on a time scale of minutes to hours.
acetyl-CoA, a signal that further oxidation of glucose for
fuel is not necessary. Acetyl-CoA is a positive allosteric Transcriptional Regulation of Glycolysis
modulator of pyruvate carboxylase and a negative
modulator of pyruvate dehydrogenase, through stimula-
and Gluconeogenesis Changes the Number
tion of a protein kinase that inactivates the dehydroge- of Enzyme Molecules
nase. When the cell’s energy needs are being met, oxi- Most of the regulatory actions discussed thus far are
dative phosphorylation slows, [NADH] rises relative to mediated by fast, quickly reversible mechanisms: alloste-
[NAD1] and inhibits the citric acid cycle, and acetyl-CoA ric effects, covalent alteration (phosphorylation) of the
enzyme, or binding of a regulatory protein. Another set
Glucose of regulatory processes involves changes in the number
of molecules of an enzyme in the cell, through changes
in the balance of enzyme synthesis and breakdown, and
our discussion now turns to regulation of transcription
Gluconeogenesis through signal-activated transcription factors.
In Chapter 12 we encountered nuclear receptors
and transcription factors in the context of insulin signal-
ing. Insulin acts through its receptor in the plasma
Oxaloacetate membrane to turn on at least two distinct signaling
pathways, each involving activation of a protein kinase.
pyruvate
carboxylase
The MAP kinase ERK, for example, phosphorylates the
transcription factors SRF and Elk1 (see Fig. 12–15),
Pyruvate which then stimulate the synthesis of enzymes needed
pyruvate
dehydrogenase
for cell growth and division. Protein kinase B (PKB; also
complex CO2 called Akt) phosphorylates another set of transcription
factors (PDX1, for example), and these stimulate the
Acetyl-CoA
synthesis of enzymes that metabolize carbohydrates
and the fats formed and stored following excess carbo-
hydrate intake in the diet. In pancreatic ␤ cells, PDX1
also stimulates the synthesis of insulin itself.
Citric acid cycle More than 150 genes are transcriptionally regulated
by insulin; humans have at least seven general types of
insulin response elements, each recognized by a subset
of transcription factors activated by insulin under vari-
Energy ous conditions. Insulin stimulates the transcription of
FIGURE 15–22 Two alternative fates for pyruvate. Pyruvate can be the genes that encode hexokinases II and IV, PFK-1,
converted to glucose and glycogen via gluconeogenesis or oxidized to pyruvate kinase, and PFK-2/FBPase-2 (all involved in
acetyl-CoA for energy production. The first enzyme in each path is regu- glycolysis and its regulation); several enzymes of fatty
lated allosterically; acetyl-CoA, produced either by fatty acid oxidation acid synthesis; and glucose 6-phosphate dehydrogenase
or by the pyruvate dehydrogenase complex, stimulates pyruvate carbox- and 6-phosphogluconate dehydrogenase, enzymes of
ylase and inhibits pyruvate dehydrogenase. the pentose phosphate pathway that generate the NADPH
8885d_c14_521-559 2/6/04 3:43 PM Page 534 mac76 mac76:385_reb:
534 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
The German biochemist Otto Warburg was the first Glucose 2NAD 2ADP 2Pi 88n
to show, as early as 1928, that tumors have a higher rate 2 pyruvate 2NADH 2H 2ATP 2H2O
of glucose metabolism than other tissues. With his as-
■ Glycolysis is tightly regulated in coordination
sociates, Warburg purified and crystallized seven of the
with other energy-yielding pathways to assure
enzymes of glycolysis. In these studies he developed and
a steady supply of ATP. Hexokinase, PFK-1,
used an experimental tool that revolutionized biochem-
and pyruvate kinase are all subject to allosteric
ical studies of oxidative metabolism: the Warburg
regulation that controls the flow of carbon
manometer, which measured directly the consumption
through the pathway and maintains constant
of oxygen by monitoring changes in gas volume, and
levels of metabolic intermediates.
therefore allowed quantitative measurement of any en-
zyme with oxidase activity.
Warburg, considered by many the preeminent bio-
chemist of the first half of the twentieth century, made
seminal contributions to many
14.2 Feeder Pathways for Glycolysis
other areas of biochemistry, Many carbohydrates besides glucose meet their cata-
including respiration, photo- bolic fate in glycolysis, after being transformed into one
synthesis, and the enzymol- of the glycolytic intermediates. The most significant are
ogy of intermediary metabo- the storage polysaccharides glycogen and starch; the
lism. Trained in carbohydrate disaccharides maltose, lactose, trehalose, and sucrose;
chemistry in the laboratory of and the monosaccharides fructose, mannose, and galac-
the great Emil Fischer (who tose (Fig. 14–9).
won the Nobel Prize in Chem-
istry in 1902), Warburg him-
self won the Nobel Prize in
Glycogen and Starch Are Degraded by Phosphorolysis
Physiology or Medicine in Glycogen in animal tissues and in microorganisms (and
Otto Warburg, 1931. A number of Warburg’s starch in plants) can be mobilized for use within the
1883–1970 students and colleagues also same cell by a phosphorolytic reaction catalyzed by
were awarded Nobel Prizes: glycogen phosphorylase (starch phosphorylase in
Otto Meyerhof in 1922, Hans Krebs and Fritz Lipmann plants). These enzymes catalyze an attack by Pi on the
in 1953, and Hugo Theorell in 1955. Meyerhof’s labora- (1n4) glycosidic linkage that joins the last two glu-
tory provided training for Lipmann, and for several other cose residues at a nonreducing end, generating glucose
Nobel Prize winners: Severo Ochoa (1959), Andre Lwoff 1-phosphate and a polymer one glucose unit shorter
(1965), and George Wald (1967). ■ (Fig. 14–10). Phosphorolysis preserves some of the en-
ergy of the glycosidic bond in the phosphate ester glu-
SUMMARY 14.1 Glycolysis cose 1-phosphate. Glycogen phosphorylase (or starch
phosphorylase) acts repetitively until it approaches an
■ Glycolysis is a near-universal pathway by which (1n6) branch point (see Fig. 7–15), where its action
a glucose molecule is oxidized to two molecules stops. A debranching enzyme removes the branches.
of pyruvate, with energy conserved as ATP and The mechanisms and control of glycogen degradation
NADH. are described in detail in Chapter 15.
■ All ten glycolytic enzymes are in the cytosol, Glucose 1-phosphate produced by glycogen phos-
and all ten intermediates are phosphorylated phorylase is converted to glucose 6-phosphate by
compounds of three or six carbons. phosphoglucomutase, which catalyzes the reversible
reaction
■ In the preparatory phase of glycolysis, ATP is
invested to convert glucose to fructose z glucose 6-phosphate
Glucose 1-phosphate y
1,6-bisphosphate. The bond between C-3 and The glucose 6-phosphate thus formed can enter glycol-
C-4 is then broken to yield two molecules of ysis or another pathway such as the pentose phosphate
triose phosphate. pathway, described in Section 14.5. Phosphoglucomu-
■ In the payoff phase, each of the two molecules tase employs essentially the same mechanism as phos-
of glyceraldehyde 3-phosphate derived from phoglycerate mutase (p. 531). The general name mu-
glucose undergoes oxidation at C-1; the energy tase is given to enzymes that catalyze the transfer of a
of this oxidation reaction is conserved in the functional group from one position to another in the
formation of one NADH and two ATP per triose same molecule. Mutases are a subclass of isomerases,
phosphate oxidized. The net equation for the enzymes that interconvert stereoisomers or structural
overall process is or positional isomers (see Table 6–3).
14.2 Feeder Pathways for Glycolysis 535
CH2OH
Trehalose Lactose O
HO H
lactase H
trehalase
OH H
H OH
H2O
CH2OH H OH
O Glycogen; starch
-amylase D-Galactose
H H
H
Pi
OH H phosphorylase
Sucrose HO OH UDP-galactose
sucrase H OH Glucose UDP-glucose

D-Glucose 1-phosphate CH2OH
ATP
hexokinase O
phosphogluco- H H
mutase H
HOCH2 CH2OH
O OH HO
Glucose
HO OH
H HO
H OH 6-phosphate
H H
ATP D-Mannose
OH H
D-Fructose hexokinase ATP
hexokinase
Fructose Mannose 6-phosphate
ATP fructokinase
6-phosphate
phosphomannose
Fructose 1-phosphate isomerase
fructose 1-
phosphate
aldolase
Fructose 1,6-
bisphosphate
Glyceraldehyde Dihydroxyacetone
phosphate
triose triose phosphate
ATP isomerase
kinase
Glyceraldehyde FIGURE 14–9 Entry of glycogen, starch, disaccharides,
3-phosphate and hexoses into the preparatory stage of glycolysis.
Dietary Polysaccharides and Disaccharides Undergo Disaccharides must be hydrolyzed to monosaccha-

Hydrolysis to Monosaccharides rides before entering cells. Intestinal disaccharides and
dextrins are hydrolyzed by enzymes attached to the
For most humans, starch is the major source of carbo-
outer surface of the intestinal epithelial cells:
hydrates in the diet. Digestion begins in the mouth,
where salivary -amylase (Fig. 14–9) hydrolyzes the in- Dextrin nH2O 8888888n n D-glucose
dextrinase
ternal glycosidic linkages of starch, producing short poly-
saccharide fragments or oligosaccharides. (Note that in Maltose H2O 8888888n 2 D-glucose
maltase
this hydrolysis reaction, water, not Pi, is the attacking
species.) In the stomach, salivary -amylase is inacti- Lactose H2O 8888888n D-galactose D-glucose
lactase
vated by the low pH, but a second form of -amylase,
secreted by the pancreas into the small intestine, con- Sucrose H2O 8888888n D-fructose D-glucose
sucrase
tinues the breakdown process. Pancreatic -amylase
yields mainly maltose and maltotriose (the di- and trisac- Trehalose H2O 8888888n 2 D-glucose
trehalase
charides of (1n4) glucose) and oligosaccharides called
limit dextrins, fragments of amylopectin containing The monosaccharides so formed are actively trans-
(1n6) branch points. Maltose and dextrins are de- ported into the epithelial cells (see Fig. 11–44), then
graded by enzymes of the intestinal brush border (the passed into the blood to be carried to various tissues,
fingerlike microvilli of intestinal epithelial cells, which where they are phosphorylated and funneled into the
greatly increase the area of the intestinal surface). Di- glycolytic sequence.
etary glycogen has essentially the same structure as Lactose intolerance, common among adults of
starch, and its digestion proceeds by the same pathway. most human populations except those originating
8885d_c14_538 2/9/04 7:04 AM Page 538 mac76 mac76:385_reb:
14.3 Fates of Pyruvate under Anaerobic Glucose
Conditions: Fermentation
Pyruvate occupies an important junction in carbohy- 2NAD
drate catabolism (Fig. 14–3). Under aerobic conditions
pyruvate is oxidized to acetate, which enters the citric 2NADH
acid cycle and is oxidized to CO2 and H2O, and NADH 2 Pyruvate 2 Lactate
formed by the dehydrogenation of glyceraldehyde 3-
The lactate formed by active skeletal muscles (or by ery-
phosphate is ultimately reoxidized to NAD by passage
throcytes) can be recycled; it is carried in the blood to
of its electrons to O2 in mitochondrial respiration. How-
the liver, where it is converted to glucose during the re-
ever, under hypoxic conditions, as in very active skele-
covery from strenuous muscular activity. When lactate
tal muscle, in submerged plant tissues, or in lactic acid
is produced in large quantities during vigorous muscle
bacteria, NADH generated by glycolysis cannot be re-
contraction (during a sprint, for example), the acidifi-
oxidized by O2. Failure to regenerate NAD would leave
cation that results from ionization of lactic acid in mus-
the cell with no electron acceptor for the oxidation of
cle and blood limits the period of vigorous activity. The
glyceraldehyde 3-phosphate, and the energy-yielding
best-conditioned athletes can sprint at top speed for no
reactions of glycolysis would stop. NAD must there-
more than a minute (Box 14–1).
fore be regenerated in some other way.
Although conversion of glucose to lactate includes
The earliest cells lived in an atmosphere almost
two oxidation-reduction steps, there is no net change in
devoid of oxygen and had to develop strategies for de-
the oxidation state of carbon; in glucose (C6H12O6) and
riving energy from fuel molecules under anaerobic
lactic acid (C3H6O3), the H:C ratio is the same. Never-
conditions. Most modern organisms have retained the
theless, some of the energy of the glucose molecule has
ability to constantly regenerate NAD during anaero-
been extracted by its conversion to lactate—enough to
bic glycolysis by transferring electrons from NADH to
give a net yield of two molecules of ATP for every glu-
form a reduced end product such as lactate or ethanol.
cose molecule consumed. Fermentation is the general
term for such processes, which extract energy (as ATP)
Pyruvate Is the Terminal Electron Acceptor in Lactic
but do not consume oxygen or change the concentra-
Acid Fermentation tions of NAD or NADH. Fermentations are carried out
When animal tissues cannot be supplied with sufficient by a wide range of organisms, many of which occupy
oxygen to support aerobic oxidation of the pyruvate and anaerobic niches, and they yield a variety of end prod-
NADH produced in glycolysis, NAD is regenerated ucts, some of which find commercial uses.
from NADH by the reduction of pyruvate to lactate. As
mentioned earlier, some tissues and cell types (such as Ethanol Is the Reduced Product in Ethanol
erythrocytes, which have no mitochondria and thus can- Fermentation
not oxidize pyruvate to CO2) produce lactate from glu-
Yeast and other microorganisms ferment glucose to
cose even under aerobic conditions. The reduction of
ethanol and CO2, rather than to lactate. Glucose is con-
pyruvate is catalyzed by lactate dehydrogenase,
verted to pyruvate by glycolysis, and the pyruvate is
which forms the L isomer of lactate at pH 7:
converted to ethanol and CO2 in a two-step process:
O O O O
NADH H
C
C O O

CO2 NADH H

NAD
C O HO C H C TPP, NAD OH
O H
lactate Mg 2
CH3 dehydrogenase
CH3 C O C CH2
pyruvate alcohol
Pyruvate L-Lactate decarboxylase dehydrogenase
CH3 CH3 CH3
G 25.1 kJ/mol Pyruvate Acetaldehyde Ethanol
The overall equilibrium of this reaction strongly favors
lactate formation, as shown by the large negative In the first step, pyruvate is decarboxylated in an irre-
standard free-energy change. versible reaction catalyzed by pyruvate decarboxy-
In glycolysis, dehydrogenation of the two molecules lase. This reaction is a simple decarboxylation and does
of glyceraldehyde 3-phosphate derived from each mol- not involve the net oxidation of pyruvate. Pyruvate de-
ecule of glucose converts two molecules of NAD to two carboxylase requires Mg2 and has a tightly bound
of NADH. Because the reduction of two molecules of coenzyme, thiamine pyrophosphate, discussed below.
pyruvate to two of lactate regenerates two molecules of In the second step, acetaldehyde is reduced to ethanol
NAD, there is no net change in NAD or NADH: through the action of alcohol dehydrogenase, with
8885d_c15_560-600 2/26/04 9:04 AM Page 576 mac76 mac76:385_reb:
576 Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen
Glucose
hexokinase glucose 6-phosphatase
Glucose 6-phosphate
phosphohexose isomerase
phospho- fructose
fructokinase-1 1,6-bisphosphatase
aldolase
Dihydroxyacetone Dihydroxyacetone
phosphate phosphate
triose phosphate triose phosphate

isomerase isomerase
Glycolysis (2) Glyceraldehyde 3-phosphate Gluconeogenesis
glyceraldehyde phosphate
dehydrogenase
(2) 1,3-Bisphosphoglycerate
phosphoglycerate kinase
phosphoglycerate mutase
enolase
(2) Phosphoenolpyruvate
PEP carboxykinase
pyruvate kinase (2) Oxaloacetate
pyruvate carboxylase
(2) Pyruvate
FIGURE 15–15 Glycolysis and gluconeogenesis. Opposing path- (the “bypass reactions”) and glycolysis; seven steps are catalyzed
ways of glycolysis (pink) and gluconeogenesis (blue) in rat liver. by the same enzymes in the two pathways. Cofactors have been
Three steps are catalyzed by different enzymes in gluconeogenesis omitted for simplicity.
allowed to proceed simultaneously at a high rate in the points of glycolysis. We then look at the regulation of
same cell, a large amount of chemical energy would be the enzymes of gluconeogenesis, leading to a consider-
dissipated as heat. This uneconomical process has been ation of how the regulation of both pathways is tightly,
called a futile cycle. However, as we shall see later, reciprocally coordinated.
such cycles may provide advantages for controlling
pathways, and the term substrate cycle is a better de- Hexokinase Isozymes of Muscle and Liver Are
scription. Similar substrate cycles also occur with the Affected Differently by Their Product, Glucose
other two sets of bypass reactions of gluconeogenesis
6-Phosphate
(Fig. 15–15).
We begin our examination of the coordinated regu- Hexokinase, which catalyzes the entry of free glucose
lation of glycolysis and gluconeogenesis by considering into the glycolytic pathway, is a regulatory enzyme.
the regulatory patterns seen at the three main control There are four isozymes (designated I to IV), encoded
TABLE 14–1 Some TPP-Dependent Reactions

Enzyme Pathway(s) Bond cleaved Bond formed
O
O O
Pyruvate decarboxylase Ethanol fermentation R1 C C R1 C
O H
O
Pyruvate dehydrogenase Synthesis of acetyl-CoA O O
-Ketoglutarate dehydrogenase Citric acid cycle R2 C C R2 C
O S-CoA
O OH O OH
Transketolase Carbon-assimilation reactions
R3 C C R4 R3 C C R5
Pentose phosphate pathway
H H
and glycerol, methanol, isopropanol, butanol, and

butanediol. These fermentations are generally carried
14.4 Gluconeogenesis
out in huge closed vats in which temperature and The central role of glucose in metabolism arose early in
access to air are controlled to favor the multiplication of evolution, and this sugar remains the nearly universal fuel
the desired microorganism and to exclude contaminat- and building block in modern organisms, from microbes to
ing organisms. The beauty of industrial fermentations is humans. In mammals, some tissues depend almost
that complicated, multistep chemical transformations completely on glucose for their metabolic energy. For the
are carried out in high yields and with few side prod- human brain and nervous system, as well as the erythro-
ucts by chemical factories that reproduce themselves— cytes, testes, renal medulla, and embryonic tissues, glucose
microbial cells. For some industrial fermentations, from the blood is the sole or major fuel source. The brain
technology has been developed to immobilize the cells alone requires about 120 g of glucose each day—more
in an inert support, to pass the starting material con- than half of all the glucose stored as glycogen in muscle
tinuously through the bed of immobilized cells, and to and liver. However, the supply of glucose from these stores
collect the desired product in the effluent—an engi- is not always sufficient; between meals and during longer
neer’s dream! fasts, or after vigorous exercise, glycogen is depleted. For
these times, organisms need a method for synthesizing
SUMMARY 14.3 Fates of Pyruvate under Anaerobic glucose from noncarbohydrate precursors. This is accom-
Conditions: Fermentation plished by a pathway called gluconeogenesis (“new
The NADH formed in glycolysis must be recycled formation of sugar”), which converts pyruvate and related
to regenerate NAD1, which is required as an three- and four-carbon compounds to glucose.
electron acceptor in the first step of the payoff Gluconeogenesis occurs in all animals, plants,
phase. Under aerobic conditions, electrons pass fungi, and microorganisms. The reactions are essen-
from NADH to O2 in mitochondrial respiration. tially the same in all tissues and all species. The impor-
tant precursors of glucose in animals are three-carbon
Under anaerobic or hypoxic conditions, many compounds such as lactate, pyruvate, and glycerol, as
organisms regenerate NAD1 by transferring well as certain amino acids (Fig. 14–16). In mammals,
electrons from NADH to pyruvate, forming lactate. gluconeogenesis takes place mainly in the liver, and to
Other organisms, such as yeast, regenerate NAD1 a lesser extent in renal cortex and in the epithelial
by reducing pyruvate to ethanol and CO2. In these cells that line the inside of the small intestine. The
anaerobic processes (fermentations), there is no glucose produced passes into the blood to supply other
net oxidation or reduction of the carbons of tissues. After vigorous exercise, lactate produced by
glucose. anaerobic glycolysis in skeletal muscle returns to the
A variety of microorganisms can ferment sugar in liver and is converted to glucose, which moves back to
fresh foods, resulting in changes in pH, taste, and muscle and is converted to glycogen—a circuit called
texture, and preserving food from spoilage. the Cori cycle (Box 14–2; see also Fig. 23–19). In plant
Fermentations are used in industry to produce a seedlings, stored fats and proteins are converted, via
wide variety of commercially valuable organic paths that include gluconeogenesis, to the disaccha-
compounds from inexpensive starting materials. ride sucrose for transport throughout the developing
14.4 Gluconeogenesis 569
Blood Other Gluconeogenesis and glycolysis are not identical

glucose Glycoproteins monosaccharides Sucrose pathways running in opposite directions, although
Glycogen Disaccharides Starch they do share several steps (Fig. 14–17); 7 of the 10
enzymatic reactions of gluconeogenesis are the reverse
of glycolytic reactions. However, three reactions of gly-
colysis are essentially irreversible in vivo and cannot
Glucose 6-phosphate
Glycolysis Gluconeogenesis
Animals Energy Plants
Glucose
ATP Pi
glucose
Phosphoenol- hexokinase
6-phosphatase
pyruvate Glucose
ADP H2O
6-phosphate
Citric
acid Fructose
cycle ATP Pi
6-phosphate
phospho- fructose
fructokinase-1 1,6-bisphosphatase-1
Pyruvate Glucogenic Glycerol 3-Phospho- Fructose
ADP H2O
amino glycerate 1,6-bisphosphate
acids
Lactate Triacyl- CO2 Dihydroxyacetone Dihydroxyacetone

glycerols fixation phosphate phosphate
(2) Glyceraldehyde
FIGURE 14–16 Carbohydrate synthesis from simple precursors. The 3-phosphate
pathway from phosphoenolpyruvate to glucose 6-phosphate is common
to the biosynthetic conversion of many different precursors of carbohy- 2Pi 2Pi
drates in animals and plants. The path from pyruvate to phosphoenol- 2NAD ⫹
2NAD⫹
pyruvate leads through oxaloacetate, an intermediate of the citric acid
2NADH ⫹ 2H⫹ 2NADH ⫹ 2H⫹
cycle, which we discuss in Chapter 16. Any compound that can be con-
verted to either pyruvate or oxaloacetate can therefore serve as starting (2) 1,3-Bisphosphoglycerate
material for gluconeogenesis. This includes alanine and aspartate, which
2ADP 2ADP
are convertible to pyruvate and oxaloacetate, respectively, and other
amino acids that can also yield three- or four-carbon fragments, the so- 2ATP 2ATP
called glucogenic amino acids (see Table 14–4; see also Fig. 18–15).
Plants and photosynthetic bacteria are uniquely able to convert CO2 to
carbohydrates, using the Calvin cycle (see Section 20.1).
plant. Glucose and its derivatives are precursors for 2GDP

(2) Phosphoenol-
the synthesis of plant cell walls, nucleotides and coen- 2ADP pyruvate PEP
carboxykinase
zymes, and a variety of other essential metabolites. In pyruvate 2GTP
many microorganisms, gluconeogenesis starts from kinase
(2) Oxaloacetate
simple organic compounds of two or three carbons, 2ATP
2ADP
such as acetate, lactate, and propionate, in their
pyruvate
growth medium. carboxylase
Although the reactions of gluconeogenesis are the 2ATP
same in all organisms, the metabolic context and the
(2) Pyruvate
regulation of the pathway differ from one species to
another and from tissue to tissue. In this section we FIGURE 14–17 Opposing pathways of glycolysis and gluconeogenesis
focus on gluconeogenesis as it occurs in the mammalian in rat liver. The reactions of glycolysis are on the left side, in red; the
liver. In Chapter 20 we show how photosynthetic organ- opposing pathway of gluconeogenesis is on the right, in blue. The major
isms use this pathway to convert the primary products sites of regulation of gluconeogenesis shown here are discussed later in
of photosynthesis into glucose, to be stored as sucrose this chapter, and in detail in Chapter 15. Figure 14–20 illustrates an alter-
or starch. native route for oxaloacetate produced in mitochondria.
14.5 Pentose Phosphate Pathway of Glucose Oxidation 549
pyruvate to PEP via oxaloacetate, catalyzed by

TABLE 14–4 Glucogenic Amino Acids, Grouped pyruvate carboxylase and PEP carboxykinase;
by Site of Entry (2) dephosphorylation of fructose
1,6-bisphosphate by FBPase-1; and
Pyruvate Succinyl-CoA
(3) dephosphorylation of glucose 6-phosphate
Alanine Isoleucine*
by glucose 6-phosphatase.
Cysteine Methionine
Glycine Threonine ■ Formation of one molecule of glucose from
Serine Valine pyruvate requires 4 ATP, 2 GTP, and 2 NADH;
Threonine it is expensive.
Fumarate
Tryptophan* Phenylalanine* ■ In mammals, gluconeogenesis in the liver and
-Ketoglutarate Tyrosine* kidney provides glucose for use by the brain,
Arginine muscles, and erythrocytes.
Oxaloacetate
Glutamate Asparagine ■ Pyruvate carboxylase is stimulated by
Glutamine Aspartate acetyl-CoA, increasing the rate of
Histidine gluconeogenesis when the cell already has
Proline adequate supplies of other substrates (fatty
acids) for energy production.
Note: All these amino acids are precursors of blood glucose or liver glycogen, because they
■ Animals cannot convert acetyl-CoA derived
can be converted to pyruvate or citric acid cycle intermediates. Of the 20 common amino
acids, only leucine and lysine are unable to furnish carbon for net glucose synthesis. from fatty acids into glucose; plants and
*These amino acids are also ketogenic (see Fig. 18–21). microorganisms can.
■ Glycolysis and gluconeogenesis are reciprocally
the result would be the consumption of ATP and the regulated to prevent wasteful operation of both
production of heat. For example, PFK-1 and FBPase-1 pathways at the same time.
catalyze opposing reactions:
ATP fructose 6-phosphate 8888888n
PFK–1 14.5 Pentose Phosphate Pathway of
ADP fructose 1,6-bisphosphate
Glucose Oxidation
Fructose 1,6-bisphosphate H2O 8888888n
FBPase–1 In most animal tissues, the major catabolic fate
fructose 6-phosphate Pi of glucose 6-phosphate is glycolytic breakdown
The sum of these two reactions is to pyruvate, much of which is then oxidized via the
citric acid cycle, ultimately leading to the formation of
ATP H2O 88n ADP Pi heat ATP. Glucose 6-phosphate does have other catabolic
These two enzymatic reactions, and a number of others fates, however, which lead to specialized products
in the two pathways, are regulated allosterically and by needed by the cell. Of particular importance in some
covalent modification (phosphorylation). In Chapter 15 tissues is the oxidation of glucose 6-phosphate to pen-
we take up the mechanisms of this regulation in detail. tose phosphates by the pentose phosphate pathway
For now, suffice it to say that the pathways are regu- (also called the phosphogluconate pathway or the
lated so that when the flux of glucose through glycoly- hexose monophosphate pathway; Fig. 14–20). In this
sis goes up, the flux of pyruvate toward glucose goes oxidative pathway, NADP is the electron acceptor,
down, and vice versa. yielding NADPH. Rapidly dividing cells, such as those of
bone marrow, skin, and intestinal mucosa, use the pen-
toses to make RNA, DNA, and such coenzymes as ATP,
SUMMARY 14.4 Gluconeogenesis NADH, FADH2, and coenzyme A.
In other tissues, the essential product of the pen-
■ Gluconeogenesis is a ubiquitous multistep tose phosphate pathway is not the pentoses but the elec-
process in which pyruvate or a related tron donor NADPH, needed for reductive biosynthesis
three-carbon compound (lactate, alanine) is or to counter the damaging effects of oxygen radicals.
converted to glucose. Seven of the steps in Tissues that carry out extensive fatty acid synthesis
gluconeogenesis are catalyzed by the same (liver, adipose, lactating mammary gland) or very ac-
enzymes used in glycolysis; these are the tive synthesis of cholesterol and steroid hormones
reversible reactions. (liver, adrenal gland, gonads) require the NADPH pro-
■ Three irreversible steps in the glycolytic vided by the pathway. Erythrocytes and the cells of
pathway are bypassed by reactions catalyzed the lens and cornea are directly exposed to oxygen and
by gluconeogenic enzymes: (1) conversion of thus to the damaging free radicals generated by oxygen.
Nonoxidative Oxidative
phase phase
HCOH
Glucose 6-phosphate A
HCOH
NADP 2 GSH A O
glutathione
HOCH
A Glucose
reductase
HCOH 6-phosphate
NADPH GSSG A
transketolase,
HC
Fatty acids, A
transaldolase 6-Phosphogluconate sterols, etc. CH2OPO2
3
NADP
reductive NADP
biosynthesis 2
glucose 6-phosphate Mg
CO2 NADPH dehydrogenase
NADPH H
Precursors
Ribulose 5-phosphate
CPO
A
HCOH
Ribose 5-phosphate A O
HOCH
A 6-Phospho-
HCOH glucono- -lactone
Nucleotides, coenzymes, A
DNA, RNA HC
A
CH2OPO23
FIGURE 14–20 General scheme of the pentose phosphate pathway.
NADPH formed in the oxidative phase is used to reduce glutathione, H 2O
lactonase
2
GSSG (see Box 14–3) and to support reductive biosynthesis. The other Mg
product of the oxidative phase is ribose 5-phosphate, which serves as
precursor for nucleotides, coenzymes, and nucleic acids. In cells that O O
M D
are not using ribose 5-phosphate for biosynthesis, the nonoxidative C
A
phase recycles six molecules of the pentose into five molecules of the HCOH
hexose glucose 6-phosphate, allowing continued production of A
HOCH 6-Phospho-
NADPH and converting glucose 6-phosphate (in six cycles) to CO2. A gluconate
HCOH
A
HCOH
By maintaining a reducing atmosphere (a high ratio of A
CH2OPO23
NADPH to NADP and a high ratio of reduced to oxi-
NADP
dized glutathione), they can prevent or undo oxidative
2
damage to proteins, lipids, and other sensitive molecules. 6-phosphogluconate Mg
In erythrocytes, the NADPH produced by the pentose dehydrogenase NADPH H
phosphate pathway is so important in preventing oxida- CO2
tive damage that a genetic defect in glucose 6-phosphate CH2OH
dehydrogenase, the first enzyme of the pathway, can A
CP O
have serious medical consequences (Box 14–3). ■ A
HCOH D-Ribulose
A
The Oxidative Phase Produces Pentose Phosphates HCOH 5-phosphate
A
and NADPH CH2OPO23
The first reaction of the pentose phosphate pathway phosphopentose
isomerase
(Fig. 14–21) is the oxidation of glucose 6-phosphate
by glucose 6-phosphate dehydrogenase (G6PD) to CHO
A
form 6-phosphoglucono--lactone, an intramolecular HCOH
ester. NADP is the electron acceptor, and the overall A
HCOH
D-Ribose
equilibrium lies far in the direction of NADPH forma- A 5-phosphate

tion. The lactone is hydrolyzed to the free acid 6-phos- HCOH
A
phogluconate by a specific lactonase, then 6-phospho- CH2OPO23
gluconate undergoes oxidation and decarboxylation by
6-phosphogluconate dehydrogenase to form the ke- FIGURE 14–21 Oxidative reactions of the pentose phosphate path-
topentose ribulose 5-phosphate. This reaction generates way. The end products are ribose 5-phosphate, CO2, and NADPH.
14.5 Pentose Phosphate Pathway of Glucose Oxidation 551
BOX 14–3 BIOCHEMISTRY IN MEDICINE
Why Pythagoras Wouldn’t Eat Falafel: Glucose The geographic distribution of G6PD deficiency is
6-Phosphate Dehydrogenase Deficiency instructive. Frequencies as high as 25% occur in trop-
ical Africa, parts of the Middle East, and Southeast
Fava beans, an ingredient of falafel, have been an im-
Asia, areas where malaria is most prevalent. In addi-
portant food source in the Mediterranean and Middle
tion to such epidemiological observations, in vitro
East since antiquity. The Greek philosopher and math-
studies show that growth of one malaria parasite, Plas-
ematician Pythagoras prohibited his followers from
modium falciparum, is inhibited in G6PD-deficient
dining on fava beans, perhaps because they make
erythrocytes. The parasite is very sensitive to oxida-
many people sick with a condition called favism, which
tive damage and is killed by a level of oxidative stress
can be fatal. In favism, erythrocytes begin to lyse 24
that is tolerable to a G6PD-deficient human host. Be-
to 48 hours after ingestion of the beans, releasing free
cause the advantage of resistance to malaria balances
hemoglobin into the blood. Jaundice and sometimes
the disadvantage of lowered resistance to oxidative
kidney failure can result. Similar symptoms can occur
damage, natural selection sustains the G6PD-deficient
with ingestion of the antimalarial drug primaquine or
genotype in human populations where malaria is
of sulfa antibiotics or following exposure to certain
prevalent. Only under overwhelming oxidative stress,
herbicides. These symptoms have a genetic basis: glu-
caused by drugs, herbicides, or divicine, does G6PD
cose 6-phosphate dehydrogenase (G6PD) deficiency,
deficiency cause serious medical problems.
which affects about 400 million people. Most G6PD-
An antimalarial drug such as primaquine is be-
deficient individuals are asymptomatic; only the com-
lieved to act by causing oxidative stress to the para-
bination of G6PD deficiency and certain environmen-
site. It is ironic that antimalarial drugs can cause ill-
tal factors produces the clinical manifestations.
ness through the same biochemical mechanism that
G6PD catalyzes the first step in the pentose phos-
provides resistance to malaria. Divicine also acts as an
phate pathway (see Fig. 14–21), which produces
antimalarial drug, and ingestion of fava beans may pro-
NADPH. This reductant, essential in many biosyn-
tect against malaria. By refusing to eat falafel, many
thetic pathways, also protects cells from oxidative
Pythagoreans with normal G6PD activity may have un-
damage by hydrogen peroxide (H2O2) and superoxide
wittingly increased their risk of malaria!
free radicals, highly reactive oxidants generated as
metabolic byproducts and through the actions of drugs
such as primaquine and natural products such as di-
vicine—the toxic ingredient of fava beans. During
normal detoxification, H2O2 is converted to H2O by re- O2
Mitochondrial respiration, ionizing
duced glutathione and glutathione peroxidase, and the radiation, sulfa drugs, herbicides,
oxidized glutathione is converted back to the reduced antimalarials, divicine
form by glutathione reductase and NADPH (Fig. 1). Superoxide O2
radical
H2O2 is also broken down to H2O and O2 by catalase, e
2H
which also requires NADPH. In G6PD-deficient
individuals, the NADPH production is diminished and Hydrogen glutathione peroxidase
H2O2 2H2O
detoxification of H2O2 is inhibited. Cellular damage peroxide
results: lipid peroxidation leading to breakdown of H e
erythrocyte membranes and oxidation of proteins H2O 2GSH GSSG
and DNA.
Hydroxyl OH
free radical
glutathione
reductase

FIGURE 1 Role of NADPH and glutathione in protecting cells Oxidative damage to NADP NADPH H
lipids, proteins, DNA
against highly reactive oxygen derivatives. Reduced glutathione
(GSH) protects the cell by destroying hydrogen peroxide and hy- Glucose 6-Phospho-
droxyl free radicals. Regeneration of GSH from its oxidized form 6-phosphate glucose glucono-d-lactone
6-phosphate
(GSSG) requires the NADPH produced in the glucose 6-phosphate dehydrogenase
dehydrogenase reaction. (G6PD)
a second molecule of NADPH. Phosphopentose iso- converted to five six-carbon sugar phosphates, com-
merase converts ribulose 5-phosphate to its aldose iso- pleting the cycle and allowing continued oxidation of
mer, ribose 5-phosphate. In some tissues, the pentose glucose 6-phosphate with production of NADPH. Con-
phosphate pathway ends at this point, and its overall tinued recycling leads ultimately to the conversion of
equation is glucose 6-phosphate to six CO2. Two enzymes unique to
the pentose phosphate pathway act in these intercon-
Glucose 6-phosphate 2NADP H2O 88n
versions of sugars: transketolase and transaldolase.
ribose 5-phosphate CO2 2NADPH 2H
Transketolase catalyzes the transfer of a two-carbon
The net result is the production of NADPH, a reductant fragment from a ketose donor to an aldose acceptor
for biosynthetic reactions, and ribose 5-phosphate, a (Fig. 14–23a). In its first appearance in the pentose
precursor for nucleotide synthesis. phosphate pathway, transketolase transfers C-1 and
C-2 of xylulose 5-phosphate to ribose 5-phosphate,
The Nonoxidative Phase Recycles Pentose forming the seven-carbon product sedoheptulose
Phosphates to Glucose 6-Phosphate 7-phosphate (Fig. 14–23b). The remaining three-carbon
fragment from xylulose is glyceraldehyde 3-phosphate.
In tissues that require primarily NADPH, the pentose Next, transaldolase catalyzes a reaction similar to
phosphates produced in the oxidative phase of the path- the aldolase reaction of glycolysis: a three-carbon frag-
way are recycled into glucose 6-phosphate. In this non- ment is removed from sedoheptulose 7-phosphate and
oxidative phase, ribulose 5-phosphate is first epimerized condensed with glyceraldehyde 3-phosphate, forming
to xylulose 5-phosphate: fructose 6-phosphate and the tetrose erythrose 4-phos-
CH2OH CH2OH phate (Fig. 14–24). Now transketolase acts again, form-
ing fructose 6-phosphate and glyceraldehyde 3-phosphate
C O C O
from erythrose 4-phosphate and xylulose 5-phosphate
H C OH HO C H (Fig. 14–25). Two molecules of glyceraldehyde 3-phos-
ribose
H C OH 5-phosphate H C OH
phate formed by two iterations of these reactions can be
epimerase converted to a molecule of fructose 1,6-bisphosphate as
CH2OPO32 CH2OPO32 in gluconeogenesis (Fig. 14–16), and finally FBPase-1 and
Ribulose Xylulose 5-phosphate phosphohexose isomerase convert fructose 1,6-bisphos-
5-phosphate
phate to glucose 6-phosphate. The cycle is complete: six
Then, in a series of rearrangements of the carbon skele- pentose phosphates have been converted to five hexose
tons (Fig. 14–22), six five-carbon sugar phosphates are phosphates (Fig. 14–22b).
oxidative reactions of
pentose phosphate pathway 5C 7C 6C
Ribose Sedoheptulose Fructose Glucose 5C 3C 4C 6C

5-phosphate 7-phosphate 6-phosphate 6-phosphate
6C
phosphohexose
epimerase 5C 3C
isomerase
transketolase transaldolase
5C 3C
Xylulose Glyceraldehyde Erythrose Fructose
5-phosphate 3-phosphate 4-phosphate 6-phosphate
fructose 1,6-
bisphosphatase
5C 3C 4C 6C
aldolase
transketolase triose phosphate
Xylulose isomerase
5-phosphate Glyceraldehyde 5C 7C 6C
3-phosphate
(a) (b)
FIGURE 14–22 Nonoxidative reactions of the pentose phosphate from six pentoses (5C) to five hexoses (6C). Note that this involves two
pathway. (a) These reactions convert pentose phosphates to hexose sets of the interconversions shown in (a). Every reaction shown here
phosphates, allowing the oxidative reactions (see Fig. 14–21) to con- is reversible; unidirectional arrows are used only to make clear the
tinue. The enzymes transketolase and transaldolase are specific to this direction of the reactions during continuous oxidation of glucose 6-
pathway; the other enzymes also serve in the glycolytic or gluco- phosphate. In the light-independent reactions of photosynthesis, the
neogenic pathways. (b) A schematic diagram showing the pathway direction of these reactions is reversed (see Fig. 20–10).
16
The Citric Acid Cycle
16.1 Production of Acetyl-CoA (Activated Acetate) 633 glycolysis and is believed to have evolved much later,
after the appearance of cyanobacteria. The metabolic
16.2 Reactions of the Citric Acid Cycle 638 activities of cyanobacteria account for the rise of oxy-
16.3 Regulation of the Citric Acid Cycle 653 gen levels in the earth’s atmosphere, a dramatic turning
point in evolutionary history.
16.4 The Glyoxylate Cycle 656 We consider first the conversion of pyruvate to acetyl
groups, then the entry of those groups into the citric acid
cycle, also called the tricarboxylic acid (TCA) cycle
A
s we saw in Chapter 14, some cells obtain energy
(ATP) by fermentation, breaking down glucose in or the Krebs cycle (after its discoverer, Hans Krebs). We
the absence of oxygen. For most eukaryotic cells next examine the cycle reactions and the enzymes that
and many bacteria, which live under aerobic conditions catalyze them. Because intermediates of the citric acid
and oxidize their organic fuels to carbon dioxide and cycle are also siphoned off as biosynthetic precursors, we
water, glycolysis is but the first stage in the complete go on to consider some ways in which these intermediates
oxidation of glucose. Rather than being reduced to lac- are replenished. The citric acid cycle is a hub in metabo-
tate, ethanol, or some other fermentation product, the lism, with degradative path-
pyruvate produced by glycolysis is further oxidized to ways leading in and anabolic
H2O and CO2. This aerobic phase of catabolism is called pathways leading out, and it is
respiration. In the broader physiological or macro- closely regulated in coordina-
scopic sense, respiration refers to a multicellular organ- tion with other pathways. The
ism’s uptake of O2 and release of CO2. Biochemists and chapter ends with a descrip-
cell biologists, however, use the term in a narrower tion of the glyoxylate path-
sense to refer to the molecular processes by which cells way, a metabolic sequence in
consume O2 and produce CO2—processes more precisely some organisms that employs
termed cellular respiration. several of the same enzymes
Cellular respiration occurs in three major stages and reactions used in the cit-
(Fig. 16–1). In the first, organic fuel molecules—glucose, ric acid cycle, bringing about
fatty acids, and some amino acids—are oxidized to yield the net synthesis of glucose Hans Krebs, 1900–1981
two-carbon fragments in the form of the acetyl group of from stored triacylglycerols.
acetyl-coenzyme A (acetyl-CoA). In the second stage,
the acetyl groups are fed into the citric acid cycle, 16.1 Production of Acetyl-CoA
which enzymatically oxidizes them to CO2; the energy
released is conserved in the reduced electron carriers
(Activated Acetate)
NADH and FADH2. In the third stage of respiration, In aerobic organisms, glucose and other sugars, fatty
these reduced coenzymes are themselves oxidized, giv- acids, and most amino acids are ultimately oxidized to
ing up protons (H1) and electrons. The electrons are CO2 and H2O via the citric acid cycle and the respiratory
transferred to O2—the final electron acceptor—via a chain. Before entering the citric acid cycle, the carbon
chain of electron-carrying molecules known as the skeletons of sugars and fatty acids are degraded to the
respiratory chain. In the course of electron transfer, the acetyl group of acetyl-CoA, the form in which the cycle
large amount of energy released is conserved in the accepts most of its fuel input. Many amino acid carbons
form of ATP, by a process called oxidative phosphoryla- also enter the cycle this way, although several amino
tion (Chapter 19). Respiration is more complex than acids are degraded to other cycle intermediates. Here
633
634 The Citric Acid Cycle
trates how a combination of covalent modification and

Stage 1 Amino Fatty allosteric mechanism results in precisely regulated flux
Acetyl-CoA acids acids Glucose
production
through a metabolic step. Finally, the PDH complex is
the prototype for two other important enzyme com-
Glycolysis
plexes: ␣-ketoglutarate dehydrogenase, of the citric
acid cycle, and the branched-chain ␣-keto acid dehy-
Pyruvate drogenase, of the oxidative pathways of several amino
e⫺
pyruvate acids (see Fig. 18–28). The remarkable similarity in the
dehydrogenase
complex
protein structure, cofactor requirements, and reaction
e⫺ mechanisms of these three complexes doubtless reflects
e⫺ CO2 a common evolutionary origin.
e⫺
Acetyl-CoA Pyruvate Is Oxidized to Acetyl-CoA and CO2
Stage 2 The overall reaction catalyzed by the pyruvate dehydro-
Acetyl-CoA genase complex is an oxidative decarboxylation, an
oxidation Citrate
Oxaloacetate irreversible oxidation process in which the carboxyl
e⫺
group is removed from pyruvate as a molecule of CO2
Citric
acid cycle and the two remaining carbons become the acetyl group
e⫺ of acetyl-CoA (Fig. 16–2). The NADH formed in this
reaction gives up a hydride ion (:H2) to the respiratory
e⫺ chain (Fig. 16–1), which carries the two electrons to
CO2 oxygen or, in anaerobic microorganisms, to an alterna-
CO2 e⫺
tive electron acceptor such as nitrate or sulfate. The
transfer of electrons from NADH to oxygen ultimately
NADH,
FADH2
generates 2.5 molecules of ATP per pair of electrons.
Stage 3 (reduced e⫺ carriers) The irreversibility of the PDH complex reaction has
Electron transfer been demonstrated by isotopic labeling experiments:
and oxidative e⫺
2H+ + 21 O2 the complex cannot reattach radioactively labeled CO2
phosphorylation
Respiratory to acetyl-CoA to yield carboxyl-labeled pyruvate.
(electron-transfer)
chain
H2O The Pyruvate Dehydrogenase Complex Requires
Five Coenzymes
ADP + Pi ATP
The combined dehydrogenation and decarboxylation of
pyruvate to the acetyl group of acetyl-CoA (Fig. 16–2)
FIGURE 16–1 Catabolism of proteins, fats, and carbohydrates in the requires the sequential action of three different enzymes
three stages of cellular respiration. Stage 1: oxidation of fatty acids, and five different coenzymes or prosthetic groups—thia-
glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation mine pyrophosphate (TPP), flavin adenine dinucleotide
of acetyl groups in the citric acid cycle includes four steps in which elec- (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to
trons are abstracted. Stage 3: electrons carried by NADH and FADH2 emphasize the role of the OSH group), nicotinamide
are funneled into a chain of mitochondrial (or, in bacteria, plasma adenine dinucleotide (NAD), and lipoate. Four different
membrane–bound) electron carriers—the respiratory chain—ultimately vitamins required in human nutrition are vital components
reducing O2 to H2O. This electron flow drives the production of ATP. of this system: thiamine (in TPP), riboflavin (in FAD),
niacin (in NAD), and pantothenate (in CoA). We have
we focus on how pyruvate, derived from glucose and
other sugars by glycolysis, is oxidized to acetyl-CoA and
CO2 by the pyruvate dehydrogenase (PDH) com- CO2
plex, a cluster of enzymes—multiple copies of each of O O ⫺ CoA-SH ⫹
M D TPP,
three enzymes—located in the mitochondria of eukary- C NAD⫹
lipoate,
NADH O S-CoA
A M D
otic cells and in the cytosol of bacteria. C PO
FAD
C
A careful examination of this enzyme complex is A pyruvate dehydrogenase A
CH3 complex (E1 ⫹ E2 ⫹ E3) CH3
rewarding in several respects. The PDH complex is a
classic, much-studied example of a multienzyme com- Pyruvate Acetyl-CoA
plex in which a series of chemical intermediates remain
⌬G⬘⬚ ⫽ ⫺33.4 kJ/mol
bound to the enzyme molecules as a substrate is trans-
formed into the final product. Five cofactors, four FIGURE 16–2 Overall reaction catalyzed by the pyruvate dehydrogenase
derived from vitamins, participate in the reaction mech- complex. The five coenzymes participating in this reaction, and the three
anism. The regulation of this enzyme complex also illus- enzymes that make up the enzyme complex, are discussed in the text.
16.1 Production of Acetyl-CoA (Activated Acetate) 635
Reactive
thiol group
NH2
N
H H H CH3 O O N Adenine
5⬘
HS CH2 CH2 N C CH2 CH2 N C C C CH2 O P O P O CH2 N N
O
␤-Mercapto- O O OH CH3 O O 4⬘ 1⬘
ethylamine H H
Pantothenic acid H H
3⬘ 2⬘ Ribose 3⬘-phosphate
O OH
O P O
O
CH3 C O
S-CoA 3⬘-Phosphoadenosine diphosphate
Coenzyme A
Acetyl-CoA
FIGURE 16–3 Coenzyme A (CoA). A hydroxyl group of pantothenic acid group not present in free ADP. The —SH group of the mercaptoethyl-
is joined to a modified ADP moiety by a phosphate ester bond, and its amine moiety forms a thioester with acetate in acetyl-coenzyme A
carboxyl group is attached to b-mercaptoethylamine in amide linkage. (acetyl-CoA) (lower left).
The hydroxyl group at the 39 position of the ADP moiety has a phosphoryl
already described the roles of FAD and NAD as electron Escherichia coli enzyme contains 24 copies of E2.) E2
carriers (Chapter 13), and we have encountered TPP as is the point of connection for the prosthetic group
the coenzyme of pyruvate decarboxylase (see Fig. 14–15). lipoate, attached through an amide bond to the ´-amino
Coenzyme A (Fig. 16–3) has a reactive thiol (—SH) group of a Lys residue (Fig. 16–4). E2 has three function-
group that is critical to the role of CoA as an acyl carrier ally distinct domains (Fig. 16–5c): the amino-terminal
in a number of metabolic reactions. Acyl groups are lipoyl domain, containing the lipoyl-Lys residue(s);
covalently linked to the thiol group, forming thioes- the central E1- and E3-binding domain; and the inner-
ters. Because of their relatively high standard free core acyltransferase domain, which contains the acyl-
energies of hydrolysis (see Figs 13–16, 13–17), thioes- transferase active site. The yeast PDH complex has a
ters have a high acyl group transfer potential and can single lipoyl domain with a lipoate attached, but the
donate their acyl groups to a variety of acceptor mole-
cules. The acyl group attached to coenzyme A may thus
be thought of as “activated” for group transfer. Oxidized Reduced Acetylated
form form O form
The fifth cofactor of the PDH complex, lipoate
CH2 HS CH2 CH3 C S CH2
(Fig. 16–4), has two thiol groups that can undergo S
CH2 CH2 CH2
reversible oxidation to a disulfide bond (—S—S—), S
CH HS CH HS CH
similar to that between two Cys residues in a protein.
CH2 CH2 CH2
Because of its capacity to undergo oxidation-reduction Lipoic
acid CH2
reactions, lipoate can serve both as an electron (hydro-
gen) carrier and as an acyl carrier, as we shall see. CH2
CH2
C O
The Pyruvate Dehydrogenase Complex Consists HN
of Three Distinct Enzymes CH2
Lys
The PDH complex contains three enzymes—pyruvate residue CH2
dehydrogenase (E1), dihydrolipoyl transacetylase of E2 CH2
(E2), and dihydrolipoyl dehydrogenase (E3)—each CH2
present in multiple copies. The number of copies of CH
each enzyme and therefore the size of the complex var- N C Polypeptide chain of
ies among species. The PDH complex isolated from H E2 (dihydrolipoyl
O
transacetylase)
mammals is about 50 nm in diameter—more than five
times the size of an entire ribosome and big enough to FIGURE 16–4 Lipoic acid (lipoate) in amide linkage with a Lys residue.
be visualized with the electron microscope (Fig. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacet-
16–5a). In the bovine enzyme, 60 identical copies of ylase (E2 of the PDH complex). The lipoyl group occurs in oxidized
E2 form a pentagonal dodecahedron (the core) with a (disulfide) and reduced (dithiol) forms and acts as a carrier of both
diameter of about 25 nm (Fig. 16–5b). (The core of the hydrogen and an acetyl (or other acyl) group.
FIGURE 16–5 The pyruvate dehydrogenase complex. (a) Cryoelectron

micrograph of PDH complexes isolated from bovine kidney. In cryoelec-
tron microscopy, biological samples are viewed at extremely low tem-
peratures; this avoids potential artifacts introduced by the usual process
of dehydrating, fixing, and staining. (b) Three-dimensional image of
PDH complex, showing the subunit structure: E1, pyruvate dehydroge-
nase; E2, dihydrolipoyl transacetylase; and E3, dihydrolipoyl dehydroge-
nase. This image is reconstructed by analysis of a large number of
images such as those in (a), combined with crystallographic studies of
individual subunits. The core (green) consists of 60 molecules of E2,
arranged in 20 trimers to form a pentagonal dodecahedron. The lipoyl
domain of E2 (blue) reaches outward to touch the active sites of E1 mol-
ecules (yellow) arranged on the E2 core. Several E3 subunits (red) are
also bound to the core, where the swinging arm on E2 can reach their
active sites. An asterisk marks the site where a lipoyl group is attached
to the lipoyl domain of E2. To make the structure clearer, about half of
the complex has been cut away from the front. This model was prepared
by Z. H. Zhou and colleagues (2001); in another model, proposed by
J. L. S. Milne and colleagues (2002), the E3 subunits are located more
toward the periphery (see Further Reading). (c) E2 consists of three
types of domains linked by short polypeptide linkers: a catalytic acyl-
transferase domain; a binding domain, involved in the binding of E2 to E1
(a) 50 nm
and E3; and one or more (depending on the species) lipoyl domains.
mammalian complex has two, and E. coli has three

(Fig. 16–5c). The domains of E2 are separated by link-
E1 ers, sequences of 20 to 30 amino acid residues, rich in
*
Ala and Pro and interspersed with charged residues;
E3 these linkers tend to assume their extended forms,
holding the three domains apart.
The active site of E1 has bound TPP, and that of E3
E2 has bound FAD. Also part of the complex are two regula-
tory proteins, a protein kinase and a phosphoprotein
phosphatase, discussed below. This basic E1–E2–E3 struc-
ture has been conserved during evolution and used in a
number of similar metabolic reactions, including the oxi-
dation of ␣-ketoglutarate in the citric acid cycle (described
below) and the oxidation of ␣-keto acids derived from the
breakdown of the branched-chain amino acids valine,
isoleucine, and leucine (see Fig. 18–28). Within a given
species, E3 of PDH is identical to E3 of the other two
10 nm enzyme complexes. The attachment of lipoate to the end
(b)
of a Lys side chain in E2 produces a long, flexible arm that
can move from the active site of E1 to the active sites of
Number of lipoyl E2 and E3, a distance of perhaps 5 nm or more.
domains varies by species.
Flexible
E. coli Mammals Yeast polypeptide
(3) (2) (1) linker In Substrate Channeling, Intermediates Never Leave
the Enzyme Surface
N C
Figure 16–6 shows schematically how the pyruvate
dehydrogenase complex carries out the five consecutive
reactions in the decarboxylation and dehydrogenation
Binding domain
(involved in E2-E1
of pyruvate. Step 1 is essentially identical to the reac-
and E2-E3 tion catalyzed by pyruvate decarboxylase (see Fig.
Lipoyl binding) 14–15c); C-1 of pyruvate is released as CO2, and C-2,
domain Acyltransferase which in pyruvate has the oxidation state of an alde-
domain hyde, is attached to TPP as a hydroxyethyl group. This
(inner core)
(c) first step is the slowest and therefore limits the rate of
16.1 Production of Acetyl-CoA (Activated Acetate) 637
O
3 CoA-SH CH3 C S- CoA
O O CH O
C Acetyl-CoA
CH3 C C
O– S Reduced
Pyruvate
3
TPP SH lipoyllysine
Acyl SH
1 2 lipoyllysine
Lys SH
TPP S
CO2 CHOH S NADH + H+
FAD
CH3
Oxidized 4
Hydroxyethyl
lipoyllysine 5
TPP FADH2
NAD+
Pyruvate Dihydrolipoyl Dihydrolipoyl

dehydrogenase, transacetylase, dehydrogenase,
E1 E2 E3
FIGURE 16–6 Oxidative decarboxylation of pyruvate to acetyl-CoA by the a transesterification in which the —SH group of CoA replaces the —SH
PDH complex. The fate of pyruvate is traced in red. In step 1 pyruvate group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the
reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydro- lipoyl group. In step 4 dihydrolipoyl dehydrogenase (E3) promotes transfer
genase (E1), undergoing decarboxylation to the hydroxyethyl derivative of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD
(see Fig. 14–15). Pyruvate dehydrogenase also carries out step 2, the prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group
transfer of two electrons and the acetyl group from TPP to the oxidized of E2. In step 5 the reduced FADH2 of E3 transfers a hydride ion to NAD1,
form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacety- forming NADH. The enzyme complex is now ready for another catalytic
lase (E2), to form the acetyl thioester of the reduced lipoyl group. Step 3 is cycle. (Subunit colors correspond to those in Fig. 16–5b.)
the overall reaction. It is also the point at which the active sites is used in some other enzymes, with lipoate,
PDH complex exercises its substrate specificity. In biotin, or a CoA-like moiety serving as cofactors.
step 2 the hydroxyethyl group is oxidized to the level As one might predict, mutations in the genes for
of a carboxylic acid (acetate). The two electrons the subunits of the PDH complex, or a dietary
removed in this reaction reduce the —S—S— of a thiamine deficiency, can have severe consequences.
lipoyl group on E2 to two thiol (—SH) groups. The Thiamine-deficient animals are unable to oxidize pyru-
acetyl moiety produced in this oxidation-reduction vate normally. This is of particular importance to the
reaction is first esterified to one of the lipoyl —SH brain, which usually obtains all its energy from the aero-
groups, then transesterified to CoA to form acetyl-CoA bic oxidation of glucose in a pathway that necessarily
(step 3). Thus the energy of oxidation drives the includes the oxidation of pyruvate. Beriberi, a disease
formation of a high-energy thioester of acetate. The that results from thiamine deficiency, is characterized
remaining reactions catalyzed by the PDH complex (by by loss of neural function. This disease occurs primarily
E3, in steps 4 and 5) are electron transfers necessary in populations that rely on a diet consisting mainly of
to regenerate the oxidized (disulfide) form of the white (polished) rice, which lacks the hulls in which
lipoyl group of E2 to prepare the enzyme complex for most of the thiamine of rice is found. People who habit-
another round of oxidation. The electrons removed ually consume large amounts of alcohol can also develop
from the hydroxyethyl group derived from pyruvate thiamine deficiency, because much of their dietary
pass through FAD to NAD1. intake consists of the vitamin-free “empty calories” of
Central to the mechanism of the PDH complex are distilled spirits. An elevated level of pyruvate in the
the swinging lipoyllysyl arms of E2, which accept from E1 blood is often an indicator of defects in pyruvate oxida-
the two electrons and the acetyl group derived from pyru- tion due to one of these causes. ■
vate, passing them to E3. All these enzymes and coen-
zymes are clustered, allowing the intermediates to react SUMMARY 16.1 Production of Acetyl-CoA
quickly without diffusing away from the surface of the (Activated Acetate)
enzyme complex. The five-reaction sequence shown in
Pyruvate, the product of glycolysis, is converted to
Figure 16–6 is thus an example of substrate channel-
acetyl-CoA, the starting material for the citric acid
ing. The intermediates of the multistep sequence never
cycle, by the pyruvate dehydrogenase complex.
leave the complex, and the local concentration of the
substrate of E2 is kept very high. Channeling also prevents The PDH complex is composed of multiple copies of
theft of the activated acetyl group by other enzymes that three enzymes: pyruvate dehydrogenase, E1 (with its
use this group as substrate. As we shall see, a similar teth- bound cofactor TPP); dihydrolipoyltransacetylase, E2
ering mechanism for the channeling of substrate between (with its covalently bound lipoyl group); and
dihydrolipoyl dehydrogenase, E3 (with its for this purpose, cells employ anaplerotic (replenishing)
cofactors FAD and NAD). reactions, which are described below.
E1 catalyzes first the decarboxylation of pyruvate, Eugene Kennedy and Albert Lehninger showed in
producing hydroxyethyl-TPP, and then the 1948 that, in eukaryotes, the entire set of reactions of
oxidation of the hydroxyethyl group to an acetyl the citric acid cycle takes place in mitochondria. Isolated
group. The electrons from this oxidation reduce mitochondria were found to contain not only all the
the disulfide of lipoate bound to E2, and the acetyl enzymes and coenzymes required for the citric acid
group is transferred into thioester linkage with one cycle, but also all the enzymes and proteins necessary
—SH group of reduced lipoate. for the last stage of respiration—electron transfer and
ATP synthesis by oxidative phosphorylation. As we shall
E2 catalyzes the transfer of the acetyl group to see in later chapters, mitochondria also contain the
coenzyme A, forming acetyl-CoA. enzymes for the oxidation of fatty acids and some amino
E3 catalyzes the regeneration of the disulfide acids to acetyl-CoA, and the oxidative degradation of
(oxidized) form of lipoate; electrons pass first to other amino acids to ␣-ketoglutarate, succinyl-CoA, or
FAD, then to NAD1. oxaloacetate. Thus, in nonphotosynthetic eukaryotes,
The long lipoyllysyl arm swings from the active site the mitochondrion is the site of most energy-yielding
of E1 to E2 to E3, tethering the intermediates to oxidative reactions and of the coupled synthesis of ATP.
the enzyme complex to allow substrate channeling. In photosynthetic eukaryotes, mitochondria are the
major site of ATP production in the dark, but in daylight
The organization of the PDH complex is very
chloroplasts produce most of the organism’s ATP. In
similar to that of the enzyme complexes that
most bacteria, the enzymes of the citric acid cycle are in
catalyze the oxidation of ␣-ketoglutarate and the
the cytosol, and the plasma membrane plays a role
branched-chain ␣-keto acids.
analogous to that of the inner mitochondrial membrane
in ATP synthesis (Chapter 19).
16.2 Reactions of the Citric Acid Cycle
We are now ready to trace the process by which acetyl-
The Sequence of Reactions in the Citric Acid Cycle
CoA undergoes oxidation. This chemical transformation Makes Chemical Sense
is carried out by the citric acid cycle, the first cyclic Acetyl-CoA produced in the breakdown of carbohy-
pathway we have encountered (Fig. 16–7). To begin a drates, fats, and proteins must be completely oxidized
turn of the cycle, acetyl-CoA donates its acetyl group to to CO2 if the maximum potential energy is to be extract-
the four-carbon compound oxaloacetate to form the ed from these fuels. However, the direct oxidation of
six-carbon citrate. Citrate is then transformed into acetate (or acetyl-CoA) to CO2 is not biochemically fea-
isocitrate, also a six-carbon molecule, which is dehydro- sible. Decarboxylation of this two-carbon acid would
genated with loss of CO2 to yield the five-carbon compound yield CO2 and methane (CH4). Methane is chemically
a-ketoglutarate (also called oxoglutarate). ␣-Ketogluta- rather stable, and except for certain methanotrophic
rate undergoes loss of a second molecule of CO2 and bacteria that grow in methane-rich niches, organisms do
ultimately yields the four-carbon compound succinate. not have the cofactors and enzymes needed to oxidize
Succinate is then enzymatically converted in three methane. Methylene groups (—CH2—), however, are
steps into the four-carbon oxaloacetate—which is then readily metabolized by enzyme systems present in most
ready to react with another molecule of acetyl-CoA. In organisms. In typical oxidation sequences, two adjacent
each turn of the cycle, one acetyl group (two carbons) methylene groups (—CH2—CH2—) are involved, at
enters as acetyl-CoA and two molecules of CO2 leave; least one of which is adjacent to a carbonyl group. As we
one molecule of oxaloacetate is used to form citrate and noted in Chapter 13 (p. 513), carbonyl groups are par-
one molecule of oxaloacetate is regenerated. No net ticularly important in the chemical transformations of
removal of oxaloacetate occurs; one molecule of oxalo- metabolic pathways. The carbon of the carbonyl group
acetate can theoretically bring about oxidation of an has a partial positive charge due to the electron-
infinite number of acetyl groups, and, in fact, oxaloace- withdrawing property of the carbonyl oxygen and is
tate is present in cells in very low concentrations. Four therefore an electrophilic center. A carbonyl group can
of the eight steps in this process are oxidations, in facilitate the formation of a carbanion on an adjoining
which the energy of oxidation is very efficiently con- carbon by delocalizing the carbanion’s negative charge.
served in the form of the reduced coenzymes NADH and We see in the citric acid cycle an example of the oxida-
FADH2. tion of a methylene group as succinate is oxidized
As noted earlier, although the citric acid cycle is (steps 6 to 8 in Fig. 16–7), forming a carbonyl (in
central to energy-yielding metabolism its role is not oxaloacetate) that is more chemically reactive than
limited to energy conservation. Four- and five-carbon either a methylene group or methane.
intermediates of the cycle serve as precursors for a wide In short, if acetyl-CoA is to be oxidized efficiently,
variety of products. To replace intermediates removed the methyl group of the acetyl-CoA must be attached to
16.2 Reactions of the Citric Acid Cycle 639
1
Claisen condensation:
Acetyl-CoA methyl group of
O acetyl-CoA converted
to methylene in citrate.
8 CH3 C S-CoA
H2O CoA-SH
Dehydrogenation:
oxidation of —OH Citrate
completes oxidation Oxaloacetate citrate synthase
sequence; generates CH2 COO⫺
Dehydration/rehydration:
carbonyl positioned to O C COO⫺ HO C COO⫺ —OH group of citrate
facilitate Claisen repositioned in isocitrate
condensation in next CH2 COO⫺ CH2 COO⫺
to set up decarboxylation
step. in next step.
malate Citric acid cycle aconitase H2O

7 dehydrogenase
Malate
Hydration: COO⫺
addition of HO CH CH2 COO⫺
water across
double bond CH2 C COO⫺
cis-Aconitate
introduces C COO⫺
COO⫺
—OH group
for next H
oxidation H2O
fumarase
step. (3) NADH
aconitase
H2O (Rehydration)
COO⫺
CH2 COO⫺
Fumarate CH
H C COO⫺
HC Isocitrate
HO C H
COO⫺ FADH2
COO ⫺ 3
isocitrate
6 succinate dehydrogenase
Oxidative
dehydrogenase decarboxylation:
Dehydrogenation: —OH group oxidized
introduction of to carbonyl, which in
double bond initiates CO2 turn facilitates
methylene oxidation CH2 COO⫺ CH2 COO⫺
a-ketoglutarate decarboxylation by
sequence. succinyl-CoA dehydrogenase CH2 stabilizing carbanion
CH2 synthetase complex formed on adjacent
Succinate COO ⫺ C O
CH2 COO⫺ carbon.
COO⫺
CH2 CoA-SH a-Ketoglutarate
CoA-SH
GTP C S-CoA CO2 4
(ATP) GDP O
5 Oxidative decarboxylation:
(ADP) Succinyl-CoA pyruvate-dehydrogenase-like
Substrate-level phosphorylation: ⫹ Pi mechanism; dependent on
energy of thioester conserved in carbonyl on adjacent carbon.
phosphoanhydride bond of GTP
or ATP.
FIGURE 16–7 Reactions of the citric acid cycle. The carbon atoms shaded in ber beside each reaction step corresponds to a numbered heading on
pink are those derived from the acetate of acetyl-CoA in the first turn of the pages 640–647). The red arrows show where energy is conserved by elec-
cycle; these are not the carbons released as CO2 in the first turn. Note that in tron transfer to FAD or NAD1, forming FADH2 or NADH 1 H1. Steps 1, 3,
succinate and fumarate, the two-carbon group derived from acetate can no and 4 are essentially irreversible in the cell; all other steps are reversible.
longer be specifically denoted; because succinate and fumarate are symmet- The nucleoside triphosphate product of step 5 may be either ATP or GTP,
ric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The num- depending on which succinyl-CoA synthetase isozyme is the catalyst.
something. The first step of the citric acid cycle neatly acetyl-CoA in a Claisen condensation (p. 513) to form
solves the problem of the unreactive methyl group by citrate (step 1 in Fig. 16–7). The methyl group of ace-
means of the condensation of acetyl-CoA with oxaloac- tate has been converted into a methylene in citric acid.
etate. The carbonyl of oxaloacetate acts as an electro- This tricarboxylic acid then readily undergoes a series
philic center, which is attacked by the methyl carbon of of oxidations that eliminate two carbons as CO2. Note
Acetyl-CoA oxidative phosphorylation (Chapter 19), passage of two

electrons from NADH to O2 drives the formation of about
2.5 ATP, and passage of two electrons from FADH2 to
Citrate
O2 yields about 1.5 ATP. This stoichiometry allows us
to calculate the overall yield of ATP from the complete
Oxaloacetate
Isocitrate oxidation of glucose. When both pyruvate molecules
are oxidized to 6 CO2 via the pyruvate dehydrogenase
NADH
Citric NADH
complex and the citric acid cycle, and the electrons are
CO2
acid transferred to O2 via oxidative phosphorylation, as
Malate cycle many as 32 ATP are obtained per glucose (Table 16–1).
a-Ketoglutarate
CO2
In round numbers, this represents the conservation of
Fumarate NADH 32 3 30.5 kJ/mol 5 976 kJ/mol or 34% of the theoretical
maximum of about 2,840 kJ/mol available from the com-
FADH2
Succinyl-CoA plete oxidation of glucose. These calculations employ
Succinate the standard free-energy changes; when corrected for
GTP the actual free energy required to form ATP within cells
(ATP)
(see Worked Example 13–2, p. 519), the calculated effi-
ciency of the process is closer to 65%.
FIGURE 16–14 Products of one turn of the citric acid cycle. At each turn
of the cycle, three NADH, one FADH2, one GTP (or ATP), and two CO2
are released in oxidative decarboxylation reactions. Here and in several Why Is the Oxidation of Acetate So Complicated?
following figures, all cycle reactions are shown as proceeding in one The eight-step cyclic process for oxidation of simple
direction only, but keep in mind that most of the reactions are reversible two-carbon acetyl groups to CO2 may seem unnecessar-
(see Fig. 16–7). ily cumbersome and not in keeping with the biological
principle of maximum economy. The role of the citric
to succinate), the four oxidation steps in the cycle acid cycle is not confined to the oxidation of acetate,
provide a large flow of electrons into the respiratory however. This pathway is the hub of intermediary
chain via NADH and FADH2 and thus lead to formation metabolism. Four- and five-carbon end products of
of a large number of ATP molecules during oxidative many catabolic processes feed into the cycle to serve as
phosphorylation. fuels. Oxaloacetate and ␣-ketoglutarate, for example,
We saw in Chapter 14 that the energy yield from the are produced from aspartate and glutamate, respec-
production of two molecules of pyruvate from one mol- tively, when proteins are degraded. Under some meta-
ecule of glucose in glycolysis is 2 ATP and 2 NADH. In bolic circumstances, intermediates are drawn out of the
TABLE 16–1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis,
the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation
Number of ATP or reduced Number of ATP
Reaction coenzyme directly formed ultimately formed*
Glucose h glucose 6-phosphate 21 ATP 21
Fructose 6-phosphate h fructose 1,6-bisphosphate 21 ATP 21
2 Glyceraldehyde 3-phosphate h 2 1,3-bisphosphoglycerate 2 NADH 3 or 5†
2 1,3-Bisphosphoglycerate h 2 3-phosphoglycerate 2 ATP 2
2 Phosphoenolpyruvate h 2 pyruvate 2 ATP 2
2 Pyruvate h 2 acetyl-CoA 2 NADH 5
2 Isocitrate h 2 a-ketoglutarate 2 NADH 5
2 ␣-Ketoglutarate h 2 succinyl-CoA 2 NADH 5
2 Succinyl-CoA h 2 succinate 2 ATP (or 2 GTP) 2
2 Succinate h 2 fumarate 2 FADH2 3
2 Malate h 2 oxaloacetate 2 NADH 5
Total 30–32
*This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2. A negative value indicates consumption.
†
This number is either 3 or 5, depending on the mechanism used to shuttle NADH equivalents from the cytosol to the mitochondrial matrix; see Figures 19–30 and 19–31.
Acetyl-CoA Citric Acid Cycle Components Are Important

PEP
or Biosynthetic Intermediates
pyruvate
In aerobic organisms, the citric acid cycle is an amphi-
CO2 bolic pathway, one that serves in both catabolic and
anabolic processes. Besides its role in the oxidative
catabolism of carbohydrates, fatty acids, and amino
acids, the cycle provides precursors for many biosyn-
Citrate thetic pathways (Fig. 16–16), through reactions that
Oxaloacetate
served the same purpose in anaerobic ancestors.
Isocitrate
␣-Ketoglutarate and oxaloacetate can, for example,
serve as precursors of the amino acids aspartate and
Malate glutamate by simple transamination (Chapter 22).
a -Ketoglutarate Through aspartate and glutamate, the carbons of oxalo-
acetate and ␣-ketoglutarate are then used to build other
Fumarate
amino acids, as well as purine and pyrimidine nucleo-
Biosynthetic tides. Oxaloacetate is converted to glucose in gluconeo-
products
Succinate
(amino acids,
genesis (see Fig. 15–13). Succinyl-CoA is a central
Succinyl-CoA nucleotides, intermediate in the synthesis of the porphyrin ring of
heme, etc.) heme groups, which serve as oxygen carriers (in hemo-
globin and myoglobin) and electron carriers (in cyto-
chromes) (see Fig. 22–25). And the citrate produced in
FIGURE 16–15 Biosynthetic precursors produced by an incomplete citric
some organisms is used commercially for a variety of
acid cycle in anaerobic bacteria. These anaerobes lack a-ketoglutarate
purposes.
dehydrogenase and therefore cannot carry out the complete citric acid
cycle. a-Ketoglutarate and succinyl-CoA serve as precursors in a variety
of biosynthetic pathways. (See Fig. 16–14 for the “normal” direction of Anaplerotic Reactions Replenish Citric Acid
these reactions in the citric acid cycle.)
Cycle Intermediates
As intermediates of the citric acid cycle are removed to
cycle to be used as precursors in a variety of biosyn- serve as biosynthetic precursors, they are replenished
thetic pathways. by anaplerotic reactions (Fig. 16–16; Table 16–2).
The citric acid cycle, like all other metabolic path- Under normal circumstances, the reactions by which
ways, is the product of evolution, and much of this evo- cycle intermediates are siphoned off into other path-
lution occurred before the advent of aerobic organisms. ways and those by which they are replenished are in
It does not necessarily represent the shortest pathway dynamic balance, so that the concentrations of the citric
from acetate to CO2, but it is the pathway that has, over acid cycle intermediates remain almost constant.
time, conferred the greatest selective advantage. Early Table 16–2 shows the most common anaplerotic reac-
anaerobes most probably used some of the reactions of tions, all of which, in various tissues and organisms, con-
the citric acid cycle in linear biosynthetic processes. In vert either pyruvate or phosphoenolpyruvate to oxaloac-
fact, some modern anaerobic microorganisms use an etate or malate. The most important anaplerotic reaction
incomplete citric acid cycle as a source of, not energy, in mammalian liver and kidney is the reversible carboxyl-
but biosynthetic precursors (Fig. 16–15). These organ- ation of pyruvate by CO2 to form oxaloacetate, catalyzed
isms use the first three reactions of the cycle to make by pyruvate carboxylase. When the citric acid cycle is
␣-ketoglutarate but, lacking ␣-ketoglutarate dehydroge- deficient in oxaloacetate or any other intermediates,
nase, they cannot carry out the complete set of citric pyruvate is carboxylated to produce more oxaloacetate.
acid cycle reactions. They do have the four enzymes The enzymatic addition of a carboxyl group to pyruvate
that catalyze the reversible conversion of oxaloacetate requires energy, which is supplied by ATP—the free
to succinyl-CoA and can produce malate, fumarate, suc- energy required to attach a carboxyl group to pyruvate is
cinate, and succinyl-CoA from oxaloacetate in a reversal about equal to the free energy available from ATP.
of the “normal” (oxidative) direction of flow through the Pyruvate carboxylase is a regulatory enzyme and is
cycle. This pathway is a fermentation, with the NADH virtually inactive in the absence of acetyl-CoA, its posi-
produced by isocitrate oxidation recycled to NAD1 by tive allosteric modulator. Whenever acetyl-CoA, the fuel
reduction of oxaloacetate to succinate. for the citric acid cycle, is present in excess, it stimu-
With the evolution of cyanobacteria that produced lates the pyruvate carboxylase reaction to produce
O2 from water, the earth’s atmosphere became aerobic more oxaloacetate, enabling the cycle to use more acetyl-
and organisms were under selective pressure to develop CoA in the citrate synthase reaction.
aerobic metabolism, which, as we have seen, is much The other anaplerotic reactions shown in Table 16–2
more efficient than anaerobic fermentation. are also regulated to keep the level of intermediates
Pyruvate
Glucose Fatty acids,

pyruvate sterols
carboxylase
Acetyl-CoA
PEP carboxykinase
Glutamine
Phosphoenolpyruvate (PEP) Oxaloacetate Citrate Proline
PEP
carboxylase Arginine
Citric acid
Malate cycle a-Ketoglutarate
Aspartate Glutamate
Serine
Asparagine
Glycine malic
enzyme
Cysteine Purines
Phenylalanine Pyrimidines Pyruvate Succinyl-CoA
Tyrosine
Tryptophan
Porphyrins,
heme
FIGURE 16–16 Role of the citric acid cycle in anabolism. Intermediates pathways. Shown in red are four anaplerotic reactions that replenish
of the citric acid cycle are drawn off as precursors in many biosynthetic depleted cycle intermediates (see Table 16–2).
high enough to support the activity of the citric acid ation reactions. It is a specialized carrier of one-carbon
cycle. Phosphoenolpyruvate (PEP) carboxylase, for groups in their most oxidized form: CO2. (The transfer
example, is activated by the glycolytic intermediate of one-carbon groups in more reduced forms is medi-
fructose 1,6-bisphosphate, which accumulates when the ated by other cofactors, notably tetrahydrofolate and
citric acid cycle operates too slowly to process the S-adenosylmethionine, as described in Chapter 18.) Car-
pyruvate generated by glycolysis. boxyl groups are activated in a reaction that consumes
ATP and joins CO2 to enzyme-bound biotin. This “acti-
vated” CO2 is then passed to an acceptor (pyruvate in
Biotin in Pyruvate Carboxylase Carries CO2 Groups this case) in a carboxylation reaction.
The pyruvate carboxylase reaction requires the vitamin Pyruvate carboxylase has four identical subunits,
biotin (Fig. 16–17), which is the prosthetic group of each containing a molecule of biotin covalently
the enzyme. Biotin plays a key role in many carboxyl- attached through an amide linkage to the -amino
TABLE 16–2 Anaplerotic Reactions

Reaction Tissue(s)/organism(s)
pyruvate carboxylase
Pyruvate 1 HCO32 1 ATP oxaloacetate 1 ADP 1 Pi Liver, kidney
PEP carboxykinase
Phosphoenolpyruvate 1 CO2 1 GDP oxaloacetate 1 GTP Heart, skeletal muscle
PEP carboxylase
Phosphoenolpyruvate 1 HCO2
3 oxaloacetate 1 Pi Higher plants, yeast, bacteria
malic enzyme
3 1 NAD(P)H
Pyruvate 1 HCO2 malate 1 NAD(P) 1 Widely distributed in eukaryotes
and bacteria
pyruvate dehydrogenase complex reaction), and the activity is turned off when ample fuel is available in the
entry of acetyl-CoA into the cycle (the citrate synthase form of fatty acids and acetyl-CoA and when the cell’s
reaction). Acetyl-CoA is also produced by pathways [ATP]/[ADP] and [NADH]/[NAD1] ratios are high, and it
other than the PDH complex reaction—most cells pro- is turned on again when energy demands are high and
duce acetyl-CoA from the oxidation of fatty acids and the cell requires greater flux of acetyl-CoA into the citric
certain amino acids—and the availability of intermedi- acid cycle.
ates from these other pathways is important in the In mammals, these allosteric regulatory mechanisms
regulation of pyruvate oxidation and of the citric acid are complemented by a second level of regulation:
cycle. The cycle is also regulated at the isocitrate covalent protein modification. The PDH complex is
dehydrogenase and ␣-ketoglutarate dehydrogenase inhibited by reversible phosphorylation of a specific Ser
reactions. residue on one of the two subunits of E1. As noted ear-
lier, in addition to the enzymes E1, E2, and E3, the mam-
Production of Acetyl-CoA by the Pyruvate malian PDH complex contains two regulatory proteins
whose sole purpose is to regulate the activity of the
Dehydrogenase Complex Is Regulated by Allosteric complex. Pyruvate dehydrogenase kinase phosphory-
and Covalent Mechanisms lates and thereby inactivates E1, and a specific phospho-
The PDH complex of mammals is strongly inhibited by protein phosphatase removes the phosphoryl group by
ATP and by acetyl-CoA and NADH, the products of the hydrolysis and thereby activates E1. The kinase is allo-
reaction catalyzed by the complex (Fig. 16–19). The sterically activated by ATP: when [ATP] is high (reflect-
allosteric inhibition of pyruvate oxidation is greatly ing a sufficient supply of energy), the PDH complex is
enhanced when long-chain fatty acids are available. inactivated by phosphorylation of E1. When [ATP]
AMP, CoA, and NAD1, all of which accumulate when too declines, kinase activity decreases and phosphatase
little acetate flows into the citric acid cycle, allosteri- action removes the phosphoryl groups from E1, activat-
cally activate the PDH complex. Thus, this enzyme ing the complex.
Pyruvate
ATP, acetyl-CoA,
pyruvate NADH, fatty acids
dehydrogenase
complex AMP, CoA, NAD, Ca2
Acetyl-CoA
NADH, succinyl-CoA, citrate, ATP

ADP
citrate
synthase Citrate
Oxaloacetate Citric
Isocitrate
acid
cycle isocitrate ATP
dehydrogenase Ca2, ADP
FIGURE 16–19 Regulation of metabolite flow from the malate
PDH complex through the citric acid cycle in mammals. dehydrogenase
NADH
The PDH complex is allosterically inhibited when [ATP]/
[ADP], [NADH]/[NAD1], and [acetyl-CoA]/[CoA] Malate
a-Ketoglutarate
ratios are high, indicating an energy-sufficient metabolic FADH2 succinyl-CoA, NADH
state. When these ratios decrease, allosteric activation of ␣ -ketoglutarate
dehydrogenase Ca2
pyruvate oxidation results. The rate of flow through the complex
citric acid cycle can be limited by the availability of the
succinate
citrate synthase substrates, oxaloacetate and acetyl-CoA, Succinyl-CoA
dehydrogenase
or of NAD1, which is depleted by its conversion to
NADH, slowing the three NAD-dependent oxidation
steps. Feedback inhibition by succinyl-CoA, citrate, and
ATP also slows the cycle by inhibiting early steps. In mus-
cle tissue, Ca21 signals contraction and, as shown here,
GTP
stimulates energy-yielding metabolism to replace the ATP (ATP)
consumed by contraction.
Several types of evidence suggest that, in cells, mul- COO COO

tienzyme complexes ensure efficient passage of the CH2 CH2
product of one enzyme reaction to the next enzyme in

the pathway. Such complexes are called metabolons. H C COO CH2
Certain enzymes of the citric acid cycle have been iso- HO C H HO C H
lated together as supramolecular complexes, or have
COO COO
been found associated with the inner mitochondrial
Isocitrate 2-Hydroxyglutarate
membrane, or have been shown to diffuse in the mito-
chondrial matrix more slowly than expected for the isocitrate dehydrogenase
individual protein in solution. There is strong evidence wild type mutant
NADP⫹
for substrate channeling through multienzyme com- NADP⫹
plexes in other metabolic pathways, and many enzymes COO NADPH ⫹ H⫹
NADPH CO2
thought of as “soluble” probably function in the cell as ⫹ H⫹ CH2
highly organized complexes that channel intermediates.
We will encounter other examples of channeling when CH2
we discuss the biosynthesis of amino acids and nucleo- C O
tides in Chapter 22.
COO
a-Ketoglutarate
Some Mutations in Enzymes of the Citric Acid Cycle
FIGURE 16-21 A mutant isocitrate dehydrogenase acquires a new
Lead to Cancer activity. Wild-type isocitrate dehydrogenase catalyzes the conversion
When the mechanisms for regulating a pathway of isocitrate to a-ketoglutarate, but mutations that alter the binding
such as the citric acid cycle are overwhelmed by site for isocitrate cause loss of the normal enzymatic activity and gain
a major metabolic perturbation, the result can be seri- of a new activity: conversion of a-ketoglutarate to 2-hydroxyglutarate.
ous disease. Mutations in citric acid cycle enzymes are Accumulation of this product inhibits histone demethylase, altering
very rare in humans and other mammals, but those that gene regulation and leading to glial cell tumors in the brain.
do occur are devastating. Genetic defects in the fuma-
rase gene lead to tumors of smooth muscle (leiomas)
and kidney; mutations in succinate dehydrogenase lead
to tumors of the adrenal gland (pheochromocytomas). SUMMARY 16.3 Regulation of the Citric Acid Cycle
In cultured cells with these mutations, fumarate (in the The overall rate of the citric acid cycle is
case of fumarase mutations) and, to a lesser extent, controlled by the rate of conversion of pyruvate to
succinate (in the case of succinate dehydrogenase acetyl-CoA and by the flux through citrate
mutations) accumulate, and this accumulation induces synthase, isocitrate dehydrogenase, and
the hypoxia-inducible transcription factor HIF-1a (see a-ketoglutarate dehydrogenase. These fluxes are
Box 14–1). The mechanism of tumor formation may be largely determined by the concentrations of
the production of a pseudohypoxic state. In cells with substrates and products: the end products ATP
these mutations, there is an up-regulation of genes nor- and NADH are inhibitory, and the substrates
mally regulated by HIF-1␣. These effects of mutations NAD1 and ADP are stimulatory.
in the fumarase and succinate dehydrogenase genes The production of acetyl-CoA for the citric acid
define them as tumor suppressor genes (p. 489). cycle by the PDH complex is inhibited
Another remarkable connection between citric allosterically by metabolites that signal a
acid cycle intermediates and cancer is the finding that sufficiency of metabolic energy (ATP, acetyl-CoA,
in many glial cell tumors (gliomas), the NADPH- NADH, and fatty acids) and stimulated by
dependent isocitrate dehydrogenase has an unusual metabolites that indicate a reduced energy supply
genetic defect. The mutant enzyme loses its normal (AMP, NAD1, CoA).
activity (converting isocitrate to a-ketoglutarate) but
gains a new activity: it converts a-ketoglutarate to
Complexes of consecutive enzymes in a pathway
2-hydroxyglutarate (Fig. 16–21), which accumulates allow substrate channeling between them.
in the tumor cells. a-Ketoglutarate and Fe31 are essen-
tial cofactors for a family of histone demethylases that
alter gene expression by removing methyl groups from
Arg and Lys residues in the histones that organize nucle-
16.4 The Glyoxylate Cycle
ar DNA. By competing with a-ketoglutarate for binding Vertebrates cannot convert fatty acids, or the acetate
to the histone demethylases, 2-hydroxyglutarate inhib- derived from them, to carbohydrates. Conversion of
its their activity. The inhibition of the histone demeth- phosphoenolpyruvate to pyruvate (p. 554) and of
ylases in turn interferes with normal gene regulation, pyruvate to acetyl-CoA (Fig. 16–2) are so exergonic as
leading to unrestricted glial cell growth. ■ to be essentially irreversible. If a cell cannot convert
16.4 The Glyoxylate Cycle 657
acetate into phosphoenolpyruvate, acetate cannot O

serve as the starting material for the gluconeogenic
CH3 C S-CoA
pathway, which leads from phosphoenolpyruvate to
Acetyl-CoA
glucose (see Fig. 15–13). Without this capacity, then,
a cell or organism is unable to convert fuels or metabo-
⫺
lites that are degraded to acetate (fatty acids and cer- O C COO
citrate
tain amino acids) into carbohydrates. CH2 COO
⫺
synthase
As noted in the discussion of anaplerotic reactions Oxaloacetate
(Table 16–2), phosphoenolpyruvate can be synthesized ⫺
CH2 COO
from oxaloacetate in the reversible reaction catalyzed NADH
⫺
by PEP carboxykinase: malate dehydrogenase
HO C COO
⫺
CH2 COO
Oxaloacetate 1 GTP ∆
NAD Citrate
phosphoenolpyruvate 1 CO2 1 GDP
COO
⫺ Glyoxylate
aconitase
Because the carbon atoms of acetate molecules that cycle
enter the citric acid cycle appear eight steps later in HO CH
oxaloacetate, it might seem that this pathway could CH2

⫺
generate oxaloacetate from acetate and thus generate ⫺ CH2 COO
COO
phosphoenolpyruvate for gluconeogenesis. However, as Malate CH COO
⫺
an examination of the stoichiometry of the citric acid malate HO CH COO

⫺
cycle shows, there is no net conversion of acetate to synthase

O
⫺
Isocitrate
oxaloacetate; in vertebrates, for every two carbons that isocitrate
C O lyase
enter the cycle as acetyl-CoA, two leave as CO2. In
O
many organisms other than vertebrates, the glyoxylate C O
cycle serves as a mechanism for converting acetate to CH3 C S-CoA H
carbohydrate. Acetyl-CoA Glyoxylate ⫺
CH2 COO
⫺
CH2 COO
The Glyoxylate Cycle Produces Four-Carbon Succinate
Compounds from Acetate FIGURE 16–22 Glyoxylate cycle. The citrate synthase, aconitase, and
In plants, certain invertebrates, and some microorgan- malate dehydrogenase of the glyoxylate cycle are isozymes of the citric
isms (including E. coli and yeast) acetate can serve both acid cycle enzymes; isocitrate lyase and malate synthase are unique to
as an energy-rich fuel and as a source of phosphoenol- the glyoxylate cycle. Notice that two acetyl groups (light red) enter the
pyruvate for carbohydrate synthesis. In these organisms, cycle and four carbons leave as succinate (blue). The glyoxylate cycle
enzymes of the glyoxylate cycle catalyze the net was elucidated by Hans Kornberg and Neil Madsen in the laboratory of
conversion of acetate to succinate or other four-carbon Hans Krebs.
intermediates of the citric acid cycle:
thus to glucose by gluconeogenesis. Vertebrates do
2 Acetyl-CoA 1 NAD 1 1 2H2O 88n
not have the enzymes specific to the glyoxylate cycle
succinate 1 2CoA 1 NADH 1 H 1
(isocitrate lyase and malate synthase) and therefore
In the glyoxylate cycle, acetyl-CoA condenses with cannot bring about the net synthesis of glucose from
oxaloacetate to form citrate, and citrate is converted lipids.
to isocitrate, exactly as in the citric acid cycle. The In plants, the enzymes of the glyoxylate cycle are
next step, however, is not the breakdown of isocitrate sequestered in membrane-bounded organelles called
by isocitrate dehydrogenase but the cleavage of isoci- glyoxysomes, which are specialized peroxisomes (Fig.
trate by isocitrate lyase, forming succinate and 16–23). Those enzymes common to the citric acid and
glyoxylate. The glyoxylate then condenses with a glyoxylate cycles have two isozymes, one specific to
second molecule of acetyl-CoA to yield malate, in a mitochondria, the other to glyoxysomes. Glyoxysomes
reaction catalyzed by malate synthase. The malate is are not present in all plant tissues at all times. They
subsequently oxidized to oxaloacetate, which can develop in lipid-rich seeds during germination, before the
condense with another molecule of acetyl-CoA to developing plant acquires the ability to make glucose by
start another turn of the cycle (Fig. 16–22). Each photosynthesis. In addition to glyoxylate cycle enzymes,
turn of the glyoxylate cycle consumes two molecules of glyoxysomes contain all the enzymes needed for the deg-
acetyl-CoA and produces one molecule of succinate, radation of the fatty acids stored in seed oils (see Fig.
which is then available for biosynthetic purposes. The 17–14). Acetyl-CoA formed from lipid breakdown is con-
succinate may be converted through fumarate and verted to succinate via the glyoxylate cycle, and the suc-
malate into oxaloacetate, which can then be converted cinate is exported to mitochondria, where citric acid
to phosphoenolpyruvate by PEP carboxykinase, and cycle enzymes transform it to malate. A cytosolic isozyme
Lipid body Lipid body
Triacylglycerols
Fatty acids
Fatty acids
Acetyl-CoA
Glyoxysome Mitochondria
FIGURE 16–23 Electron micrograph of a germinating cucumber seed,

Oxaloacetate
showing a glyoxysome, mitochondria, and surrounding lipid bodies.
Glyoxylate
Malate cycle Citrate
Sucrose
of malate dehydrogenase oxidizes malate to oxaloace-
tate, a precursor for gluconeogenesis. Germinating Glyoxylate
seeds can therefore convert the carbon of stored lipids Acetyl-CoA Isocitrate
into glucose. Hexoses
Succinate
Glyoxysome gluconeogenesis
The Citric Acid and Glyoxylate Cycles Are Oxaloacetate
Coordinately Regulated
Cytosol Malate
In germinating seeds, the enzymatic transformations of
dicarboxylic and tricarboxylic acids occur in three intra-
cellular compartments: mitochondria, glyoxysomes, and
the cytosol. There is a continuous interchange of metab-
olites among these compartments (Fig. 16–24).
The carbon skeleton of oxaloacetate from the citric
acid cycle (in the mitochondrion) is carried to the glyoxy-
Fumarate
some in the form of aspartate. Aspartate is converted to Malate
oxaloacetate, which condenses with acetyl-CoA derived
Citric
from fatty acid breakdown. The citrate thus formed is acid
converted to isocitrate by aconitase, then split into glyox- Succinate cycle Oxaloacetate
ylate and succinate by isocitrate lyase. The succinate
returns to the mitochondrion, where it reenters the citric
acid cycle and is transformed into malate, which enters Citrate
the cytosol and is oxidized (by cytosolic malate dehydro-
genase) to oxaloacetate. Oxaloacetate is converted via Mitochondrion
gluconeogenesis into hexoses and sucrose, which can be
transported to the growing roots and shoot. Four distinct
FIGURE 16–24 Relationship between the glyoxylate and citric acid cycles.
pathways participate in these conversions: fatty acid The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultane-
breakdown to acetyl-CoA (in glyoxysomes), the glyoxyl- ously with, and mesh with, those of the citric acid cycle (in mitochondria),
ate cycle (in glyoxysomes), the citric acid cycle (in mito- as intermediates pass between these compartments. The conversion of
chondria), and gluconeogenesis (in the cytosol). succinate to oxaloacetate is catalyzed by citric acid cycle enzymes. The
The sharing of common intermediates requires that oxidation of fatty acids to acetyl-CoA is described in Chapter 17; the syn-
these pathways be coordinately regulated. Isocitrate is thesis of hexoses from oxaloacetate is described in Chapter 20.
a crucial intermediate, at the branch point between the
glyoxylate and citric acid cycles (Fig. 16–25). Isoci-
trate dehydrogenase is regulated by covalent modifica- citric acid cycle. The regulatory protein kinase and
tion: a specific protein kinase phosphorylates and phosphoprotein phosphatase are separate enzymatic
thereby inactivates the dehydrogenase. This inactiva- activities of a single polypeptide.
tion shunts isocitrate to the glyoxylate cycle, where it Some bacteria, including E. coli, have the full com-
begins the synthetic route toward glucose. A phospho- plement of enzymes for the glyoxylate and citric acid
protein phosphatase removes the phosphoryl group cycles in the cytosol and can therefore grow on acetate as
from isocitrate dehydrogenase, reactivating the enzyme their sole source of carbon and energy. The phosphopro-
and sending more isocitrate through the energy-yielding tein phosphatase that activates isocitrate dehydrogenase
Further Reading 659
Acetyl-CoA SUMMARY 16.4 The Glyoxylate Cycle

intermediates intermediates
of citric acid of citric acid
The glyoxylate cycle is active in the germinating
cycle and cycle and seeds of some plants and in certain microorganisms
glycolysis, glycolysis,
AMP, ADP Isocitrate AMP, ADP
that can live on acetate as the sole carbon source.
In plants, the pathway takes place in glyoxysomes
in seedlings. It involves several citric acid cycle
protein enzymes and two additional enzymes: isocitrate
isocitrate
lyase
kinase lyase and malate synthase.
isocitrate
dehydrogenase phosphatase In the glyoxylate cycle, the bypassing of the two
decarboxylation steps of the citric acid cycle
makes possible the net formation of succinate,
Succinate,
a-Ketoglutarate oxaloacetate, and other cycle intermediates from
glyoxylate
acetyl-CoA. Oxaloacetate thus formed can be used
Glyoxylate Citric acid to synthesize glucose via gluconeogenesis.
cycle cycle
Vertebrates lack the glyoxylate cycle and cannot
synthesize glucose from acetate or the fatty acids
that give rise to acetyl-CoA.
The partitioning of isocitrate between the citric
acid cycle and the glyoxylate cycle is controlled at
Oxaloacetate NADH,
FADH2 the level of isocitrate dehydrogenase, which is
gluconeogenesis
oxidative
regulated by reversible phosphorylation.
phosphorylation
Glucose
ATP
Amino acids,
Key Terms
nucleotides Terms in bold are defined in the glossary.
FIGURE 16–25 Coordinated regulation of glyoxylate and citric acid respiration 633 nucleoside diphosphate
cycles. Regulation of isocitrate dehydrogenase activity determines the cellular respiration 633 kinase 645
partitioning of isocitrate between the glyoxylate and citric acid cycles. citric acid cycle 633 synthases 646
When the enzyme is inactivated by phosphorylation (by a specific protein tricarboxylic acid (TCA) synthetases 646
kinase), isocitrate is directed into biosynthetic reactions via the glyoxylate cycle 633 ligases 646
cycle. When the enzyme is activated by dephosphorylation (by a specific Krebs cycle 633 lyases 646
phosphatase), isocitrate enters the citric acid cycle and ATP is produced. pyruvate dehydrogenase kinases 646
(PDH) complex 634 phosphorylases 646
oxidative phosphatases 646
is stimulated by intermediates of the citric acid cycle and decarboxylation 634 prochiral molecule 648
glycolysis and by indicators of reduced cellular energy thioester 635 amphibolic
supply (Fig. 16–25). The same metabolites inhibit the lipoate 635 pathway 650
protein kinase activity of the bifunctional polypeptide. substrate channeling 637 anaplerotic
Thus, the accumulation of intermediates of the central iron-sulfur center 641 reaction 650
energy-yielding pathways—indicating energy depletion— moonlighting biotin 651
results in the activation of isocitrate dehydrogenase. enzymes 642 avidin 653
When the concentration of these regulators falls, signaling ␣-ketoglutarate metabolon 656
a sufficient flux through the energy-yielding citric acid dehydrogenase glyoxylate cycle 657
cycle, isocitrate dehydrogenase is inactivated by the pro- complex 644
tein kinase.
The same intermediates of glycolysis and the citric
acid cycle that activate isocitrate dehydrogenase are
allosteric inhibitors of isocitrate lyase. When energy- Further Reading
yielding metabolism is sufficiently fast to keep the
concentrations of glycolytic and citric acid cycle inter- General
mediates low, isocitrate dehydrogenase is inactivated, Holmes, F.L. (1990, 1993) Hans Krebs, Vol 1: Formation of a
Scientific Life, 1900–1933; Vol. 2: Architect of Intermediary
the inhibition of isocitrate lyase is relieved, and isocitrate
Metabolism, 1933–1937, Oxford University Press, Oxford.
flows into the glyoxylate pathway, to be used in the A scientific and personal biography of Krebs by an eminent
biosynthesis of carbohydrates, amino acids, and other historian of science, with a thorough description of the work that
cellular components. revealed the urea and citric acid cycles.
8885d_c15_562 2/26/04 2:52 PM Page 562 mac76 mac76:385_reb:
15.1 The Metabolism of Glycogen amount stored as fat (triacylglycerol) (see Table 23–5),
but fats cannot be converted to glucose in mammals and
in Animals cannot be catabolized anaerobically.
In a wide range of organisms, excess glucose is con- Glycogen granules are complex aggregates of glyco-
verted to polymeric forms for storage—glycogen in ver- gen and the enzymes that synthesize it and degrade it,
tebrates and many microorganisms, starch in plants. In as well as the machinery for regulating these enzymes.
vertebrates, glycogen is found primarily in the liver and The general mechanisms for storing and mobilizing
skeletal muscle; it may represent up to 10% of the glycogen are the same in muscle and liver, but the en-
weight of liver and 1% to 2% of the weight of muscle. zymes differ in subtle yet important ways that reflect
If this much glucose were dissolved in the cytosol of a the different roles of glycogen in the two tissues. Glyco-
hepatocyte, its concentration would be about 0.4 M, gen is also obtained in the diet and broken down in the
enough to dominate the osmotic properties of the cell. gut, and this involves a separate set of hydrolytic
When stored as a long polymer (glycogen), however, the enzymes that convert glycogen (and starch) to free
same mass of glucose has a concentration of only glucose.
0.01 M. Glycogen is stored in large cytosolic granules. The transformations of glucose discussed in this
The elementary particle of glycogen, the -particle, about chapter and in Chapter 14 are central to the metabo-
21 nm in diameter, consists of up to 55,000 glucose lism of most organisms, microbial, animal, or plant. We
residues with about 2,000 nonreducing ends. Twenty to begin with a discussion of the catabolic pathways from
40 of these particles cluster together to form -rosettes, glycogen to glucose 6-phosphate (glycogenolysis) and
easily seen with the microscope in tissue samples from from glucose 6-phosphate to pyruvate (glycolysis),
well-fed animals (Fig. 15–2) but essentially absent after then turn to the anabolic pathways from pyruvate to
a 24-hour fast. glucose (gluconeogenesis) and from glucose to glyco-
The glycogen in muscle is there to provide a quick gen (glycogenesis).
source of energy for either aerobic or anaerobic metab-
olism. Muscle glycogen can be exhausted in less than an Glycogen Breakdown Is Catalyzed
hour during vigorous activity. Liver glycogen serves as by Glycogen Phosphorylase
a reservoir of glucose for other tissues when dietary glu-
In skeletal muscle and liver, the glucose units of the
cose is not available (between meals or during a fast);
outer branches of glycogen enter the glycolytic pathway
this is especially important for the neurons of the brain,
through the action of three enzymes: glycogen phos-
which cannot use fatty acids as fuel. Liver glycogen can
phorylase, glycogen debranching enzyme, and phos-
be depleted in 12 to 24 hours. In humans, the total
phoglucomutase. Glycogen phosphorylase catalyzes the
amount of energy stored as glycogen is far less than the
reaction in which an (1n4) glycosidic linkage between
two glucose residues at a nonreducing end of glycogen
undergoes attack by inorganic phosphate (Pi ), remov-
ing the terminal glucose residue as -D-glucose 1-phos-
phate (Fig. 15–3). This phosphorolysis reaction is
different from the hydrolysis of glycosidic bonds by
amylase during intestinal degradation of dietary glyco-
gen and starch. In phosphorolysis, some of the energy
of the glycosidic bond is preserved in the formation of
the phosphate ester, glucose 1-phosphate.
Pyridoxal phosphate is an essential cofactor in the
glycogen phosphorylase reaction; its phosphate group
acts as a general acid catalyst, promoting attack by Pi
on the glycosidic bond. (This is an unusual role for this
cofactor; its more typical role is as a cofactor in amino
acid metabolism; see Fig. 18–6.)
Glycogen phosphorylase acts repetitively on the
nonreducing ends of glycogen branches until it reaches
FIGURE 15–2 Glycogen granules in a hepatocyte. Glycogen is a stor- a point four glucose residues away from an (1n6)
age form of carbohydrate in cells, especially hepatocytes, as illustrated branch point (see Fig. 7–15), where its action stops.
here. Glycogen appears as electron-dense particles, often in aggre- Further degradation by glycogen phosphorylase can oc-
gates or rosettes. In hepatocytes the glycogen is closely associated with cur only after the debranching enzyme, formally
tubules of the smooth endoplasmic reticulum. Many mitochondria are known as oligo (1n6) to (1n4) glucantrans-
also present. ferase, catalyzes two successive reactions that transfer
15.1 The Metabolism of Glycogen in Animals 563
FIGURE 15–3 Removal of a terminal Nonreducing end

glucose residue from the nonreducing 6 CH
2OH CH2OH CH2OH
end of a glycogen chain by glycogen 5 O O O
phosphorylase. This process is repeti- H H H H H H
H H H
tive; the enzyme removes successive 4 1
glucose residues until it reaches the OH H OH H OH H
HO O O OO
fourth glucose unit from a branch point 3 2
(see Fig. 15–4). H OH H OH H OH Glycogen chain
(glucose)n
Pi glycogen
phosphorylase
Nonreducing end
6 CH
2OH CH2OH CH2OH
5 O O O
H H H H H H
H H H
4 1 O
OH H A OH H OH H
HO O OP OO HO O OO
3 2 B
H OH O H OH H OH
Glucose 1-phosphate Glycogen shortened
by one residue
(glucose)n1
Nonreducing branches (Fig. 15–4). Once these branches are trans-

ends ferred and the glucosyl residue at C-6 is hydrolyzed,
(α1→6)
linkage glycogen phosphorylase activity can continue.
Glucose 1-Phosphate Can Enter Glycolysis or,

in Liver, Replenish Blood Glucose
Glycogen
Glucose 1-phosphate, the end product of the glycogen
glycogen phosphorylase reaction, is converted to glucose 6-phos-
phosphorylase phate by phosphoglucomutase, which catalyzes the
reversible reaction
z glucose 6-phosphate
Initially phosphorylated at a Ser residue, the enzyme do-

Glucose 1-phosphate nates a phosphoryl group to C-6 of the substrate, then
molecules
accepts a phosphoryl group from C-1 (Fig. 15–5).
transferase
activity of
The glucose 6-phosphate formed from glycogen in
debranching skeletal muscle can enter glycolysis and serve as an en-
enzyme
ergy source to support muscle contraction. In liver,
FIGURE 15–4 Glycogen breakdown near an (1n6) branch point.

(α1→6) Following sequential removal of terminal glucose residues by glyco-
glucosidase gen phosphorylase (see Fig. 15–3), glucose residues near a branch are
activity of
debranching
removed in a two-step process that requires a bifunctional “de-
enzyme Glucose branching enzyme.” First, the transferase activity of the enzyme shifts
a block of three glucose residues from the branch to a nearby nonre-
ducing end, to which they are reattached in (1n4) linkage. The sin-
gle glucose residue remaining at the branch point, in (1n6) linkage,
is then released as free glucose by the enzyme’s (1n6) glucosidase
Unbranched (α1→4) polymer;
substrate for further activity. The glucose residues are shown in shorthand form, which
phosphorylase action omits the OH, OOH, and OCH2OH groups from the pyranose rings.
FIGURE 15–5 Reaction catalyzed by phosphogluco- HOCH2

mutase. The reaction begins with the enzyme O Phosphoglucomutase
phosphorylated on a Ser residue. In step 1 , the H H H
O O
enzyme donates its phosphoryl group (green) to OH H
glucose 1-phosphate, producing glucose 1,6-bisphos- HO O P O– Ser O P O–
phate. In step 2 , the phosphoryl group at C-1 of
H HO O– O–
glucose 1,6-bisphosphate (red) is transferred back to
Glucose 1-phosphate
the enzyme, re-forming the phosphoenzyme and
producing glucose 6-phosphate. 1
O
–O CH2
P O
–O O
H H H
O
OH H
HO O P O– Ser OH
H HO O–
Glucose 1,6-bisphosphate
O 2
–O CH2
P O
O– O
H H H
OH H
HO OH
H HO
Glucose 6-phosphate
glycogen breakdown serves a different purpose: to re- with its active site on the lumenal side of the ER. Glu-
lease glucose into the blood when the blood glucose cose 6-phosphate formed in the cytosol is transported
level drops, as it does between meals. This requires an into the ER lumen by a specific transporter (T1) (Fig.
enzyme, glucose 6-phosphatase, that is present in liver 15–6) and hydrolyzed at the lumenal surface by the glu-
and kidney but not in other tissues. The enzyme is an cose 6-phosphatase. The resulting Pi and glucose are
integral membrane protein of the endoplasmic reticu- thought to be carried back into the cytosol by two dif-
lum, predicted to contain nine transmembrane helices, ferent transporters (T2 and T3), and the glucose leaves
Cytosol Plasma
membrane
Glucose
G6P 6-phosphatase
G6P Capillary
Glucose
transporter
transporter
(T1)
(T2)
G6P Glucose Glucose
GLUT2
ER Pi Pi
lumen
Pi transporter
(T3)
Increased
blood
glucose
concentration
FIGURE 15–6 Hydrolysis of glucose 6-phosphate by glucose 6- ucts glucose and Pi pass to the cytosol on specific transporters (T2 and
phosphatase of the ER. The catalytic site of glucose 6-phosphatase T3). Glucose leaves the cell via the GLUT2 transporter in the plasma
faces the lumen of the ER. A glucose 6-phosphate (G6P) transporter membrane.
(T1) carries the substrate from the cytosol to the lumen, and the prod-
the hepatocyte via yet another transporter in the plasma D- Glucosyl group
membrane (GLUT2). Notice that by having the active CH2OH
site of glucose 6-phosphatase inside the ER lumen, the O
H H H
cell separates this reaction from the process of glycol-
ysis, which takes place in the cytosol and would be OH H Uridine
HO
aborted by the action of glucose 6-phosphatase. Genetic O
defects in either glucose 6-phosphatase or T1 lead to O O
H HO
HN
serious derangement of glycogen metabolism, resulting O P O P O
in type Ia glycogen storage disease (Box 15–1). O
O O CH2 N
Because muscle and adipose tissue lack glucose
O
6-phosphatase, they cannot convert the glucose 6-
phosphate formed by glycogen breakdown to glucose, H H
and these tissues therefore do not contribute glucose to H H
the blood. OH OH
UDP-glucose
The Sugar Nucleotide UDP-Glucose Donates Glucose (a sugar nucleotide)
for Glycogen Synthesis
Many of the reactions in which hexoses are transformed The suitability of sugar nucleotides for biosynthetic
or polymerized involve sugar nucleotides, compounds reactions stems from several properties:
in which the anomeric carbon of a sugar is activated by
attachment to a nucleotide through a phosphate ester 1. Their formation is metabolically irreversible, con-
linkage. Sugar nucleotides are the substrates for poly- tributing to the irreversibility of the synthetic
merization of monosaccharides into disaccharides, pathways in which they are intermediates. The
glycogen, starch, cellulose, and more complex extracel- condensation of a nucleoside triphosphate with a
lular polysaccharides. They are also key intermediates hexose 1-phosphate to form a sugar nucleotide
in the production of the aminohexoses and deoxyhex- has a small positive free-energy change, but the
oses found in some of these polysaccharides, and in the reaction releases PPi, which is rapidly hydrolyzed
synthesis of vitamin C (L-ascorbic acid). The role of by inorganic pyrophosphatase in a reaction that is
sugar nucleotides in the biosynthesis of glycogen and strongly exergonic (G 19.2 kJ/mol; Fig.
many other carbohydrate derivatives was first discov- 15–7). This keeps the cellular concentration of PPi
ered by the Argentine biochemist Luis Leloir. low, ensuring that the actual free-energy change in
O O O O

Sugar O P O O P O P O P O Ribose Base

O O O O
Sugar phosphate NTP
NDP-sugar
pyrophosphorylase
O O O O

O P O P O Sugar O P O P O Ribose Base
Luis Leloir, 1906–1987

O O O O
Pyrophosphate (PPi) Sugar nucleotide
(NDP-sugar)
inorganic
pyrophosphatase
FIGURE 15–7 Formation of a sugar nucleotide. A
O
condensation reaction occurs between a nucleoside

triphosphate (NTP) and a sugar phosphate. The negatively 2 O P OH
charged oxygen on the sugar phosphate serves as a
O
nucleophile, attacking the phosphate of the nucleoside
Phosphate (Pi)
triphosphate and displacing pyrophosphate. The reaction
is pulled in the forward direction by the hydrolysis of PPi
by inorganic pyrophosphatase. Net reaction: Sugar phosphate NTP NDP-sugar 2Pi
polysaccharide synthesis, 1970), Earl Sutherland (for cellular fractionation, 1974), and Edwin Krebs (for the
the discovery of cAMP in the regulation of carbohy- discovery of phosphorylase kinase, 1991).
drate metabolism, 1971), Christian de Duve (for sub-
TABLE 1 Glycogen Storage Diseases of Humans

Primary organ
Type (name) Enzyme affected affected Symptoms
Type 0 Glycogen synthase Liver Low blood glucose, high
ketone bodies, early death
Type Ia (von Gierke’s) Glucose 6-phosphatase Liver Enlarged liver, kidney failure
Type Ib Microsomal glucose Liver As in Ia; also high
6-phosphate translocase susceptibility to bacterial
infections
Type Ic Microsomal Pi Liver As in Ia
transporter
Type II (Pompe’s) Lysosomal glucosidase Skeletal and Infantile form: death by age 2;
cardiac muscle juvenile form: muscle defects
(myopathy); adult form: as in
muscular dystrophy
Type IIIa (Cori’s or Forbes’s) Debranching enzyme Liver, skeletal Enlarged liver in infants;
and cardiac myopathy
muscle
Type IIIb Liver debranching Liver Enlarged liver in infants
enzyme (muscle
enzyme normal)
Type IV (Andersen’s) Branching enzyme Liver, skeletal Enlarged liver and spleen,
muscle myoglobin in urine
Type V (McArdle’s) Muscle phosphorylase Skeletal muscle Exercise-induced cramps and
pain; myoglobin in urine
Type VI (Hers’s) Liver phosphorylase Liver Enlarged liver
Type VII (Tarui’s) Muscle PFK-1 Muscle, As in V; also hemolytic
erythrocytes anemia
Type VIb, VIII, or IX Phosphorylase kinase Liver, leukocytes, Enlarged liver
muscle
Type XI (Fanconi-Bickel) Glucose transporter Liver Failure to thrive, enlarged
(GLUT2) liver, rickets, kidney
dysfunction
4. By “tagging” some hexoses with nucleotidyl is glucose 6-phosphate. As we saw in Chapter 14, this
groups, cells can set them aside in a pool for one can be derived from free glucose in a reaction catalyzed
purpose (glycogen synthesis, for example), sepa- by the isozymes hexokinase I and hexokinase II in
rate from hexose phosphates destined for another muscle and hexokinase IV (glucokinase) in liver:
purpose (such as glycolysis). D-Glucose ATP 88n D-glucose 6-phosphate ADP
Glycogen synthesis takes place in virtually all animal However, some ingested glucose takes a more roundabout
tissues but is especially prominent in the liver and skele- path to glycogen. It is first taken up by erythrocytes and
tal muscles. The starting point for synthesis of glycogen converted to lactate glycolytically; the lactate is then
taken up by the liver and converted to glucose 6-phos- gen molecule (Fig. 15–8). The overall equilibrium of the
phate by gluconeogenesis. path from glucose 6-phosphate to lengthened glycogen
To initiate glycogen synthesis, the glucose 6- greatly favors synthesis of glycogen.
phosphate is converted to glucose 1-phosphate in the Glycogen synthase cannot make the (1n6) bonds
phosphoglucomutase reaction: found at the branch points of glycogen; these are formed
z glucose 1-phosphate by the glycogen-branching enzyme, also called amylo
(1n4) to (1n6) transglycosylase or glycosyl-
The product of this reaction is converted to UDP- (4n6)-transferase. The glycogen-branching enzyme
glucose by the action of UDP-glucose pyrophosphor- catalyzes transfer of a terminal fragment of 6 or 7 glu-
ylase, in a key step of glycogen biosynthesis: cose residues from the nonreducing end of a glycogen
branch having at least 11 residues to the C-6 hydroxyl
Glucose 1-phosphate UTP 88n UDP-glucose PPi
group of a glucose residue at a more interior position of
Notice that this enzyme is named for the reverse reaction; the same or another glycogen chain, thus creating a new
in the cell, the reaction proceeds in the direction of UDP- branch (Fig. 15–9). Further glucose residues may be
glucose formation, because pyrophosphate is rapidly added to the new branch by glycogen synthase. The
hydrolyzed by inorganic pyrophosphatase (Fig. 15–7). biological effect of branching is to make the glycogen
UDP-glucose is the immediate donor of glucose res- molecule more soluble and to increase the number of
idues in the reaction catalyzed by glycogen synthase, nonreducing ends. This increases the number of sites
which promotes the transfer of the glucose residue from accessible to glycogen phosphorylase and glycogen syn-
UDP-glucose to a nonreducing end of a branched glyco- thase, both of which act only at nonreducing ends.
6 CH
2OH
5
O
H H
H
4 1
OH H
HO O
3 2
H HO
O

O P O P O
CH2OH CH2OH
O O CH2
Uracil O O
O H H H H
H H
UDP-glucose 4 1 4 1
H H OH H OH H
H H HO O O
OH OH H OH H OH
Nonreducing end of
a glycogen chain
glycogen
synthase with n residues
UDP (n 4)
CH2OH CH2OH CH2OH

O O O
H H H H H H
New nonreducing H H H
4 1 4 1 4 1
end OH H OH H OH H
HO O O O
H OH H OH H OH
Elongated glycogen
with n 1 residues
FIGURE 15–8 Glycogen synthesis. A glycogen chain is elongated by glycogen synthase. The en-
zyme transfers the glucose residue of UDP-glucose to the nonreducing end of a glycogen branch
(see Fig. 7–15) to make a new (1n4) linkage.
O O O O O O O O O O O
HO O O O O O O O O O O
Glycogen
core
Nonreducing ( 1 4)
end glycogen-branching
enzyme
O O O O O O O
Nonreducing
HO O O O O O O
end
O ( 1 6) Branch
O O O O
point
Nonreducing HO Glycogen
O O O
end core
FIGURE 15–9 Branch synthesis in glycogen. The glycogen-branching enzyme (also called amylo
(1n4) to (1n6) transglycosylase or glycosyl-(4n6)-transferase) forms a new branch point during
glycogen synthesis.
Glycogenin Primes the Initial Sugar Residues cule is the transfer of a glucose residue from UDP-
glucose to the hydroxyl group of Tyr194 of glycogenin,
in Glycogen
catalyzed by the protein’s intrinsic glucosyltransferase
Glycogen synthase cannot initiate a new glycogen chain activity (Fig. 15–11a). The nascent chain is extended by
de novo. It requires a primer, usually a preformed the sequential addition of seven more glucose residues,
(1n4) polyglucose chain or branch having at least each derived from UDP-glucose; the reactions are cat-
eight glucose residues. How is a new glycogen molecule alyzed by the chain-extending activity of glycogenin. At
initiated? The intriguing protein glycogenin (Fig. this point, glycogen synthase takes over, further ex-
15–10) is both the primer on which new chains are as- tending the glycogen chain. Glycogenin remains buried
sembled and the enzyme that catalyzes their assembly. within the particle, covalently attached to the single re-
The first step in the synthesis of a new glycogen mole- ducing end of the glycogen molecule (Fig. 15–11b).
FIGURE 15–10 Glycogenin structure. (PDB 1D 1772)

Muscle glycogenin (Mr 37,000) forms dimers in
solution. Humans have a second isoform in liver,
glycogenin-2. The substrate, UDP-glucose (shown as a
red ball-and-stick structure), is bound to a Rossman fold
near the amino terminus and is some distance from the
Tyr194 residues (turquoise)—15 Å from that in the same
monomer, 12 Å from that in the dimeric partner. Each
UDP-glucose is bound through its phosphates to a
Mn2 ion (green) that is essential to catalysis. Mn2 is
believed to function as an electron-pair acceptor (Lewis
acid) to stabilize the leaving group, UDP. The glycosidic
bond in the product has the same configuration about
the C-1 of glucose as the substrate UDP-glucose,
suggesting that the transfer of glucose from UDP to
194
Tyr occurs in two steps. The first step is probably a
nucleophilic attack by Asp162 (orange), forming a
temporary intermediate with inverted configuration. A
second nucleophilic attack by Tyr194 then restores the
starting configuration.
(a) (b)
CH2OH Each chain has

O 12 to 14 glucose
:
H H H HO Glycogenin residues
OH H
HO O Tyr194
H HO
O
UDP-glucose –O
P O P O–
O O Ribose Uracil
glucosyltransferase
activity
G
UDP-glucose UDP
CH2OH
O
H H
CH2OH O G glycogenin third tier
OH H
O HO
:
H H primer fourth tier

H
H HO outer tier
OH H second tier
HO O (unbranched)
H HO
O
UDP-glucose MECHANISM FIGURE 15–11 Glycogenin and the structure of the glycogen
–O
P O P O– particle. (a) Glycogenin catalyzes two distinct reactions. Initial attack by the
O O Ribose Uracil hydroxyl group of Tyr194 on C-1 of the glucosyl moiety of UDP-glucose results
in a glucosylated Tyr residue. The C-1 of another UDP-glucose molecule is
now attacked by the C-4 hydroxyl group of the terminal glucose, and this
chain-extending
activity sequence repeats to form a nascent glycogen molecule of eight glucose
residues attached by (1n4) glycosidic linkages. (b) Structure of the glycogen
UDP particle. Starting at a central glycogenin molecule, glycogen chains (12 to 14
residues) extend in tiers. Inner chains have two ( 1n6) branches each. Chains
in the outer tier are unbranched. There are 12 tiers in a mature glycogen
Repeats six times particle (only 5 are shown here), consisting of about 55,000 glucose residues
in a molecule of about 21 nm diameter and Mr 107.
SUMMARY 15.1 The Metabolism of Glycogen 6-phosphate can enter glycolysis or, in liver,
in Animals can be converted to free glucose by glucose
6-phosphatase in the endoplasmic reticulum,
■ Glycogen is stored in muscle and liver as large then released to replenish blood glucose.
particles. Contained within the particles are the ■ The sugar nucleotide UDP-glucose donates
enzymes that metabolize glycogen, as well as glucose residues to the nonreducing end of
regulatory enzymes. glycogen in the reaction catalyzed by glycogen
■ Glycogen phosphorylase catalyzes synthase. A separate branching enzyme
phosphorolytic cleavage at the nonreducing produces the (1n6) linkages at branch points.
ends of glycogen chains, producing glucose ■ New glycogen particles begin with the auto-
1-phosphate. The debranching enzyme transfers catalytic formation of a glycosidic bond between
branches onto main chains and releases the the glucose of UDP-glucose and a Tyr residue
residue at the (1n6) branch as free glucose. in the protein glycogenin, followed by addition
■ Phosphoglucomutase interconverts glucose of several glucose residues to form a primer
1-phosphate and glucose 6-phosphate. Glucose that can be acted upon by glycogen synthase.
19.1 Electron-Transfer Reactions in Mitochondria 691
processes involve the flow of electrons through a chain ATP synthase Outer membrane
of membrane-bound carriers. (2) The free energy made (FoF1)
Freely permeable to
available by this “downhill” (exergonic) electron flow Cristae small molecules and ions
is coupled to the “uphill” transport of protons across a
proton-impermeable membrane, conserving the free
energy of fuel oxidation as a transmembrane electro- Inner membrane
chemical potential (p. 391). (3) The transmembrane
Impermeable to most
flow of protons down their concentration gradient small molecules and ions,
through specific protein channels provides the free including H
energy for synthesis of ATP, catalyzed by a membrane Contains:
protein complex (ATP synthase) that couples proton • Respiratory electron
carriers (Complexes I–IV)
flow to phosphorylation of ADP.
• ADP-ATP translocase
We begin this chapter with oxidative phosphoryla-
• ATP synthase (FoF1)
tion. We first describe the components of the electron-
• Other membrane
transfer chain, their organization into large functional
transporters
complexes in the inner mitochondrial membrane, the
path of electron flow through them, and the proton
movements that accompany this flow. We then consider Matrix
the remarkable enzyme complex that, by “rotational Contains:
catalysis,” captures the energy of proton flow in ATP, • Pyruvate
and the regulatory mechanisms that coordinate oxida- dehydrogenase
tive phosphorylation with the many catabolic pathways complex
by which fuels are oxidized. With this understanding of • Citric acid
cycle enzymes
mitochondrial oxidative phosphorylation, we turn to
• Fatty acid
photophosphorylation, looking first at the absorption of -oxidation
light by photosynthetic pigments, then at the light- enzymes
driven flow of electrons from H2O to NADP and the • Amino acid
molecular basis for coupling electron and proton flow. oxidation
enzymes
We also consider the similarities of structure and mech-
Ribosomes • DNA, ribosomes
anism between the ATP synthases of chloroplasts and
• Many other enzymes
mitochondria, and the evolutionary basis for this con- Porin channels
• ATP, ADP, Pi, Mg2, Ca2, K
servation of mechanism.
• Many soluble metabolic
intermediates
OXIDATIVE PHOSPHORYLATION
FIGURE 19–1 Biochemical anatomy of a mitochondrion. The convo-
19.1 Electron-Transfer Reactions lutions (cristae) of the inner membrane provide a very large surface
in Mitochondria area. The inner membrane of a single liver mitochondrion may have
more than 10,000 sets of electron-transfer systems (respiratory chains)
The discovery in 1948 by Eugene Kennedy and Albert and ATP synthase molecules, distributed over the membrane surface.
Lehninger that mitochondria are the site of oxidative Heart mitochondria, which have more profuse cristae and thus a much
phosphorylation in eukaryotes marked the beginning larger area of inner membrane, contain more than three times as many
of the modern phase of studies sets of electron-transfer systems as liver mitochondria. The mitochon-
in biological energy transduc- drial pool of coenzymes and intermediates is functionally separate from
tions. Mitochondria, like gram- the cytosolic pool. The mitochondria of invertebrates, plants, and mi-
negative bacteria, have two crobial eukaryotes are similar to those shown here, but with much vari-
membranes (Fig. 19–1). The ation in size, shape, and degree of convolution of the inner membrane.
outer mitochondrial membrane
is readily permeable to small
molecules (Mr 5,000) and molecules and ions, including protons (H); the only
ions, which move freely species that cross this membrane do so through specific
through transmembrane chan- transporters. The inner membrane bears the compo-
nels formed by a family of inte- nents of the respiratory chain and the ATP synthase.
gral membrane proteins called The mitochondrial matrix, enclosed by the inner
Albert L. Lehninger, porins. The inner membrane is membrane, contains the pyruvate dehydrogenase com-
1917–1986 impermeable to most small plex and the enzymes of the citric acid cycle, the fatty
8885d_c19_696 3/1/04 1:58 PM Page 696 mac76 mac76:385_reb:
696 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
rotenone FIGURE 19–6 Method for determining the

sequence of electron carriers. This method
NADH Q Cyt b Cyt c1 Cyt c Cyt (a a3) O2 measures the effects of inhibitors of electron
transfer on the oxidation state of each carrier.
In the presence of an electron donor and O2,
antimycin A each inhibitor causes a characteristic pattern
of oxidized/reduced carriers: those before the
NADH Q Cyt b Cyt c1 Cyt c Cyt ( a a3) O2 block become reduced (blue), and those after
the block become oxidized (pink).
CN or CO
NADH Q Cyt b Cyt c1 Cyt c Cyt (a a3) O2
of electron carriers involves reducing the entire chain complexes that can be physically separated. Gentle
of carriers experimentally by providing an electron treatment of the inner mitochondrial membrane with
source but no electron acceptor (no O2). When O2 is detergents allows the resolution of four unique electron-
suddenly introduced into the system, the rate at which carrier complexes, each capable of catalyzing electron
each electron carrier becomes oxidized (measured transfer through a portion of the chain (Table 19–3; Fig.
spectroscopically) reveals the order in which the car- 19–7). Complexes I and II catalyze electron transfer to
riers function. The carrier nearest O2 (at the end of the ubiquinone from two different electron donors: NADH
chain) gives up its electrons first, the second carrier (Complex I) and succinate (Complex II). Complex III
from the end is oxidized next, and so on. Such exper- carries electrons from reduced ubiquinone to cyto-
iments have confirmed the sequence deduced from chrome c, and Complex IV completes the sequence by
standard reduction potentials. transferring electrons from cytochrome c to O2.
In a final confirmation, agents that inhibit the flow We now look in more detail at the structure and
of electrons through the chain have been used in com- function of each complex of the mitochondrial respira-
bination with measurements of the degree of oxidation tory chain.
of each carrier. In the presence of O2 and an electron
donor, carriers that function before the inhibited step Complex I: NADH to Ubiquinone Figure 19–8 illustrates the
become fully reduced, and those that function after this relationship between Complexes I and II and ubiquinone.
step are completely oxidized (Fig. 19–6). By using sev- Complex I, also called NADH:ubiquinone oxidore-
eral inhibitors that block different steps in the chain, in- ductase or NADH dehydrogenase, is a large enzyme
vestigators have determined the entire sequence; it is composed of 42 different polypeptide chains, including
the same as deduced in the first two approaches. an FMN-containing flavoprotein and at least six iron-
sulfur centers. High-resolution electron microscopy
shows Complex I to be L-shaped, with one arm of the L
Electron Carriers Function in Multienzyme Complexes
in the membrane and the other extending into the ma-
The electron carriers of the respiratory chain are or- trix. As shown in Figure 19–9, Complex I catalyzes two
ganized into membrane-embedded supramolecular simultaneous and obligately coupled processes: (1) the
TABLE 19–3 The Protein Components of the Mitochondrial Electron-Transfer Chain

Enzyme complex/protein Mass (kDa) Number of subunits* Prosthetic group(s)
I NADH dehydrogenase 850 43 (14) FMN, Fe-S
II Succinate dehydrogenase 140 4 FAD, Fe-S
III Ubiquinone cytochrome c oxidoreductase 250 11 Hemes, Fe-S
Cytochrome c† 13 1 Heme
IV Cytochrome oxidase 160 13 (3–4) Hemes; CuA, CuB
*
Numbers of subunits in the bacterial equivalents in parentheses.
†
Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein.
Amytal (a barbiturate drug), rotenone (a plant product

4H+
commonly used as an insecticide), and piericidin A (an
Complex I Intermembrane antibiotic) inhibit electron flow from the Fe-S centers
space (P side) of Complex I to ubiquinone (Table 19–4) and therefore
block the overall process of oxidative phosphorylation.
Q
2e– Ubiquinol (QH2, the fully reduced form; Fig. 19–2)
N-2 QH2 diffuses in the inner mitochondrial membrane from
Fe-S Complex I to Complex III, where it is oxidized to Q in a
process that also involves the outward movement of H.
Matrix FMN Matrix (N side)
arm Complex II: Succinate to Ubiquinone We encountered
2e–
2H+ Membrane
Complex II in Chapter 16 as succinate dehydroge-
arm nase, the only membrane-bound enzyme in the citric
acid cycle (p. 612). Although smaller and simpler than
NADH NAD+ H+ Complex I, it contains five prosthetic groups of two
types and four different protein subunits (Fig. 19–10).
FIGURE 19–9 NADH:ubiquinone oxidoreductase (Complex I). Com-
Subunits C and D are integral membrane proteins, each
plex I catalyzes the transfer of a hydride ion from NADH to FMN, from
with three transmembrane helices. They contain a heme
which two electrons pass through a series of Fe-S centers to the iron-
sulfur protein N-2 in the matrix arm of the complex. Electron transfer
group, heme b, and a binding site for ubiquinone, the
from N-2 to ubiquinone on the membrane arm forms QH2, which dif-
final electron acceptor in the reaction catalyzed by
fuses into the lipid bilayer. This electron transfer also drives the ex- Complex II. Subunits A and B extend into the matrix (or
pulsion from the matrix of four protons per pair of electrons. The de- the cytosol of a bacterium); they contain three 2Fe-2S
tailed mechanism that couples electron and proton transfer in Complex centers, bound FAD, and a binding site for the substrate,
I is not yet known, but probably involves a Q cycle similar to that in succinate. The path of electron transfer from the
Complex III in which QH2 participates twice per electron pair (see succinate-binding site to FAD, then through the Fe-S
Fig. 19–12). Proton flux produces an electrochemical potential across centers to the Q-binding site, is more than 40 Å long,
the inner mitochondrial membrane (N side negative, P side positive), but none of the individual electron-transfer distances
which conserves some of the energy released by the electron-transfer exceeds about 11 Å—a reasonable distance for rapid
reactions. This electrochemical potential drives ATP synthesis. electron transfer (Fig. 19–10).
TABLE 19–4 Agents That Interfere with Oxidative Phosphorylation or Photophosphorylation

Type of interference Compound* Target/mode of action
Inhibition of electron transfer Cyanide 
 Inhibit cytochrome oxidase
Carbon monoxide 
Antimycin A Blocks electron transfer from cytochrome b to cytochrome c1
Myxothiazol 
Rotenone 
 Prevent electron transfer from Fe-S center to ubiquinone
Amytal 
Piericidin A 
DCMU Competes with QB for binding site in PSII
Inhibition of ATP synthase Aurovertin Inhibits F1
Oligomycin 
 Inhibit Fo and CFo
Venturicidin 
DCCD Blocks proton flow through Fo and CFo
Uncoupling of phosphorylation FCCP 
 Hydrophobic proton carriers
from electron transfer DNP 
Valinomycin K ionophore
Thermogenin In brown fat, forms proton-conducting pores in inner mitochondrial
membrane
Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase
*
DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-trifluoromethoxyphenylhydrazone; DNP,
2,4-dinitrophenol.
Plant Mitochondria Have Alternative Mechanisms Complex IV, cytochrome oxidase. This
for Oxidizing NADH copper-containing enzyme, which also contains
cytochromes a and a3, accumulates electrons,
Plant mitochondria supply the cell with ATP during then passes them to O2, reducing it to H2O.
periods of low illumination or darkness by mechanisms
■ Some electrons enter this chain of carriers
entirely analogous to those used by nonphotosynthetic
through alternative paths. Succinate is oxidized
organisms. In the light, the principal source of mito-
by succinate dehydrogenase (Complex II),
chondrial NADH is a reaction in which glycine, produced
which contains a flavoprotein that passes
by a process known as photorespiration, is converted to
electrons through several Fe-S centers to
serine (see Fig. 20–21):
ubiquinone. Electrons derived from the
2 Glycine NAD 88n serine CO2 NH3 NADH H oxidation of fatty acids pass to ubiquinone via
For reasons discussed in Chapter 20, plants must carry the electron-transferring flavoprotein.
out this reaction even when they do not need NADH for ■ Plants also have an alternative, cyanide-resistant
ATP production. To regenerate NAD from unneeded NADH oxidation pathway.
NADH, plant mitochondria transfer electrons from NADH
directly to ubiquinone and from ubiquinone directly to
O2, bypassing Complexes III and IV and their proton 19.2 ATP Synthesis
pumps. In this process the energy in NADH is dissipated How is a concentration gradient of protons transformed
as heat, which can sometimes be of value to the plant into ATP? We have seen that electron transfer releases,
(Box 19–1). Unlike cytochrome oxidase (Complex IV), and the proton-motive force conserves, more than
the alternative QH2 oxidase is not inhibited by cyanide. enough free energy (about 200 kJ) per “mole” of elec-
Cyanide-resistant NADH oxidation is therefore the hall- tron pairs to drive the formation
mark of this unique plant electron-transfer pathway. of a mole of ATP, which requires
about 50 kJ (see Box 13–1). Mi-
SUMMARY 19.1 Electron-Transfer Reactions tochondrial oxidative phospho-
in Mitochondria rylation therefore poses no
thermodynamic problem. But
■ Chemiosmotic theory provides the intellectual what is the chemical mechanism
framework for understanding many biological that couples proton flux with
energy transductions, including oxidative phosphorylation?
phosphorylation and photophosphorylation. The chemiosmotic model,
The mechanism of energy coupling is similar in proposed by Peter Mitchell, is
both cases: the energy of electron flow is the paradigm for this mecha-
conserved by the concomitant pumping of nism. According to the model Peter Mitchell,
protons across the membrane, producing an (Fig. 19–17), the electrochemi- 1920–1992
electrochemical gradient, the proton-motive cal energy inherent in the difference in proton concen-
force. tration and separation of charge across the inner mito-
■ In mitochondria, hydride ions removed from chondrial membrane—the proton-motive force—drives
substrates by NAD-linked dehydrogenases the synthesis of ATP as protons flow passively back into
donate electrons to the respiratory the matrix through a proton pore associated with ATP
(electron-transfer) chain, which transfers the synthase. To emphasize this crucial role of the proton-
electrons to molecular O2, reducing it to H2O. motive force, the equation for ATP synthesis is some-
times written
■ Shuttle systems convey reducing equivalents
from cytosolic NADH to mitochondrial NADH. ADP Pi nH
P On ATP H2O nH N (19–10)
Reducing equivalents from all NAD-linked Mitchell used “chemiosmotic” to describe enzymatic re-
dehydrogenations are transferred to mito- actions that involve, simultaneously, a chemical reaction
chondrial NADH dehydrogenase (Complex I). and a transport process. The operational definition of
■ Reducing equivalents are then passed through “coupling” is shown in Figure 19–18. When isolated mi-
a series of Fe-S centers to ubiquinone, which tochondria are suspended in a buffer containing ADP,
transfers the electrons to cytochrome b, the Pi, and an oxidizable substrate such as succinate, three
first carrier in Complex III. In this complex, easily measured processes occur: (1) the substrate is
electrons take two separate paths through two oxidized (succinate yields fumarate), (2) O2 is consumed,
b-type cytochromes and cytochrome c1 to an and (3) ATP is synthesized. Oxygen consumption and
Fe-S center. The Fe-S center passes electrons, ATP synthesis depend on the presence of an oxidizable
one at a time, through cytochrome c and into substrate (succinate in this case) as well as ADP and Pi.
19.2 ATP Synthesis 705
FIGURE 19–17 Chemiosmotic model. In this

2H+ simple representation of the chemiosmotic
4H+ theory applied to mitochondria, electrons from
4H+ Cyt c NADH and other oxidizable substrates pass
Intermembrane
+ + + + + through a chain of carriers arranged asymmet-
space +
+ IV + rically in the inner membrane. Electron flow is
+
Q accompanied by proton transfer across the
III +
I II – + + membrane, producing both a chemical
– +
– H2 O + + gradient (pH) and an electrical gradient ().
– 1
Fumarate O + 2H+
2 2 – The inner mitochondrial membrane is imper-
Succinate +
– + meable to protons; protons can reenter the
ADP + Pi
NADH + H+ NAD+ Fo matrix only through proton-specific channels
–
+ – (Fo). The proton-motive force that drives
Matrix F1
ATP – protons back into the matrix provides the
H+ – energy for ATP synthesis, catalyzed by the F1
Chemical ATP Electrical
potential synthesis potential – – complex associated with Fo.
–
∆pΗ driven by ∆w – –
(inside proton-motive (inside
alkaline) force negative)
Because the energy of substrate oxidation drives hibitors of both ATP synthesis and the transfer of elec-
ATP synthesis in mitochondria, we would expect in- trons through the chain of carriers to O2 (Fig. 19–18b).
hibitors of the passage of electrons to O2 (such as Because oligomycin is known to interact not directly with
cyanide, carbon monoxide, and antimycin A) to block the electron carriers but with ATP synthase, it follows
ATP synthesis (Fig. 19–18a). More surprising is the find- that electron transfer and ATP synthesis are obligately
ing that the converse is also true: inhibition of ATP syn- coupled; neither reaction occurs without the other.
thesis blocks electron transfer in intact mitochondria. Chemiosmotic theory readily explains the depend-
This obligatory coupling can be demonstrated in isolated ence of electron transfer on ATP synthesis in mitochon-
mitochondria by providing O2 and oxidizable substrates, dria. When the flow of protons into the matrix through
but not ADP (Fig. 19–18b). Under these conditions, no the proton channel of ATP synthase is blocked (with
ATP synthesis can occur and electron transfer to O2 oligomycin, for example), no path exists for the return
does not proceed. Coupling of oxidation and phosphor- of protons to the matrix, and the continued extrusion
ylation can also be demonstrated using oligomycin or of protons driven by the activity of the respiratory chain
venturicidin, toxic antibiotics that bind to the ATP syn- generates a large proton gradient. The proton-motive
thase in mitochondria. These compounds are potent in- force builds up until the cost (free energy) of pumping
Add Add Add

CN venturicidin DNP
or Uncoupled
ATP synthesized
oligomycin
ATP synthesized
O2 consumed
O2 consumed
Add Add
succinate ADP Pi
Add Add
ADP Pi succinate
(a) Time (b) Time
FIGURE 19–18 Coupling of electron transfer and ATP synthesis in ATP is synthesized. Addition of cyanide (CN), which blocks electron
mitochondria. In experiments to demonstrate coupling, mitochondria transfer between cytochrome oxidase and O2, inhibits both respiration
are suspended in a buffered medium and an O2 electrode monitors O2 and ATP synthesis. (b) Mitochondria provided with succinate respire
consumption. At intervals, samples are removed and assayed for the and synthesize ATP only when ADP and Pi are added. Subsequent ad-
presence of ATP. (a) Addition of ADP and Pi alone results in little or no dition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks
increase in either respiration (O2 consumption; black) or ATP synthe- both ATP synthesis and respiration. Dinitrophenol (DNP) is an un-
sis (red). When succinate is added, respiration begins immediately and coupler, allowing respiration to continue without ATP synthesis.
18.1 Metabolic Fates of Amino Groups 703
glutaminase converts glutamine to glutamate and NH41 Muscle

(Fig. 18–8). The NH41 from intestine and kidney is protein
transported in the blood to the liver. In the liver, the
ammonia from all sources is disposed of by urea synthe- Amino acids
sis. Some of the glutamate produced in the glutaminase Muscle
reaction may be further processed in the liver by gluta-
⫹
NH4
mate dehydrogenase, releasing more ammonia and Glucose Pyruvate
producing carbon skeletons for metabolic fuel. However, glycolysis Glutamate
most glutamate enters the transamination reactions alanine
required for amino acid biosynthesis and other processes aminotransferase
(Chapter 22). ␣-Ketoglutarate
In metabolic acidosis (p. 688) there is an increase Alanine
in glutamine processing by the kidneys. Not all
the excess NH41 thus produced is released into the
Blood
bloodstream or converted to urea; some is excreted Blood alanine
directly into the urine. In the kidney, the NH41 forms glucose
salts with metabolic acids, facilitating their removal in
Liver
the urine. Bicarbonate produced by the decarboxylation Alanine
␣-Ketoglutarate
of ␣-ketoglutarate in the citric acid cycle can also serve
as a buffer in blood plasma. Taken together, these alanine
aminotransferase
effects of glutamine metabolism in the kidney tend to
counteract acidosis. ■ Glutamate
Glucose Pyruvate
gluconeogenesis
Alanine Transports Ammonia from Skeletal Muscles NH⫹
4
to the Liver
urea cycle
Alanine also plays a special role in transporting amino
groups to the liver in a nontoxic form, via a pathway Urea
called the glucose-alanine cycle (Fig. 18–9). In
muscle and certain other tissues that degrade amino FIGURE 18–9 Glucose-alanine cycle. Alanine serves as a carrier of
acids for fuel, amino groups are collected in the form of ammonia and of the carbon skeleton of pyruvate from skeletal muscle to
glutamate by transamination (Fig. 18–2a). Glutamate liver. The ammonia is excreted and the pyruvate is used to produce
can be converted to glutamine for transport to the liver, glucose, which is returned to the muscle.
as described above, or it can transfer its ␣-amino group
to pyruvate, a readily available product of muscle gly-
on the liver rather than the muscle, and all available ATP
colysis, by the action of alanine aminotransferase
in muscle is devoted to muscle contraction.
(Fig. 18–9). The alanine so formed passes into the blood
and travels to the liver. In the cytosol of hepatocytes,
alanine aminotransferase transfers the amino group Ammonia Is Toxic to Animals
from alanine to ␣-ketoglutarate, forming pyruvate and The catabolic production of ammonia poses a
glutamate. Glutamate can then enter mitochondria, serious biochemical problem, because ammonia
where the glutamate dehydrogenase reaction releases is very toxic. The molecular basis for this toxicity is not
NH41 (Fig. 18–7), or can undergo transamination with entirely understood. The terminal stages of ammonia
oxaloacetate to form aspartate, another nitrogen donor intoxication in humans are characterized by onset of a
in urea synthesis, as we shall see. comatose state accompanied by cerebral edema (an
The use of alanine to transport ammonia from skel- increase in the brain’s water content) and increased
etal muscles to the liver is another example of the intrin- cranial pressure, so research and speculation on ammo-
sic economy of living organisms. Vigorously contracting nia toxicity have focused on this tissue. Speculation
skeletal muscles operate anaerobically, producing pyru- centers on a potential depletion of ATP in brain cells.
vate and lactate from glycolysis as well as ammonia from Ammonia readily crosses the blood-brain barrier,
protein breakdown. These products must find their way so any condition that raises the level of ammonia in
to the liver, where pyruvate and lactate are incorporated the bloodstream will expose the brain to high concen-
into glucose, which is returned to the muscles, and trations too. The developing brain is more susceptible
ammonia is converted to urea for excretion. The to the deleterious effects of ammonium ion than the
glucose-alanine cycle, in concert with the Cori cycle (see adult brain. The damage from ammonium toxicity
Box 14–2 and Fig. 23–19), accomplishes this transaction. includes loss of neurons, altered synapse formation,
The energetic burden of gluconeogenesis is thus imposed and a general defect in cellular energy metabolism.
948 Hormonal Regulation and Integration of Mammalian Metabolism
(concentration of the compound) PCr Actively contracting skeletal muscle generates heat
as a byproduct of imperfect coupling of the chemical
energy of ATP with the mechanical work of contraction.
Intensity of signal
This heat production can be put to good use when ambi-

Pi ATP ent temperature is low: skeletal muscle carries out shiv-
ering thermogenesis, rapidly repeated muscle con-
Recovery
traction that produces heat but little motion, helping to
maintain the body at its preferred temperature of 37 ºC.
Exercise
Heart muscle differs from skeletal muscle in that
Rest
it is continuously active in a regular rhythm of
0 –5 –10 –15 –20
Chemical shift (parts per million)
contraction and relaxation and it has a completely aero-
(identity of the compound) bic metabolism at all times. Mitochondria are much more
abundant in heart muscle than in skeletal muscle, mak-
FIGURE 23–18 Phosphocreatine buffers ATP concentration during exer-
ing up almost half the volume of the cells (Fig. 23–20).
cise. A ”stack plot” of magnetic resonance spectra (of 31P) showing inor-
The heart uses mainly free fatty acids, but also some
ganic phosphate (Pi), phosphocreatine (PCr), and ATP (each of its three
phosphates giving a signal). The series of plots represents the passage of
glucose and ketone bodies taken up from the blood, as
time, from a period of rest to one of exercise, and then of recovery. Note
sources of energy; these fuels are oxidized via the citric
that the ATP signal hardly changes during exercise, kept high by contin- acid cycle and oxidative phosphorylation to generate
ued respiration and by the reservoir of phosphocreatine, which diminishes ATP. Like skeletal muscle, heart muscle does not store
during exercise. During recovery, when ATP production by catabolism is lipids or glycogen in large amounts. It does have small
greater than ATP utilization by the (now resting) muscle, the phosphocre- amounts of reserve energy in the form of phosphocre-
atine reservoir is refilled. atine, enough for a few seconds of contraction. Because
the heart is normally aerobic and obtains its energy from
After a period of intense muscular activity, the indi- oxidative phosphorylation, the failure of O2 to reach part
vidual continues breathing heavily for some time, using of the heart muscle when the blood vessels are blocked
much of the extra O2 for oxidative phosphorylation in by lipid deposits (atherosclerosis) or blood clots (coro-
the liver. The ATP produced is used for gluconeogenesis nary thrombosis) can cause that region of the heart
(in the liver) from lactate that has been carried in the muscle to die. This is what happens in myocardial infarc-
blood from the muscles. The glucose thus formed tion, more commonly known as a heart attack. ■
returns to the muscles to replenish their glycogen, com-
pleting the Cori cycle (Fig. 23–19; see also Box 15–4).
The Brain Uses Energy for Transmission
of Electrical Impulses
The metabolism of the brain is remarkable in several
Muscle: ATP produced by respects. The neurons of the adult mammalian brain
glycolysis for rapid contraction.
Lactate Glycogen
Mitochondria Actin-myosin
contractile apparatus
ATP
Blood
Blood glucose
lactate
ATP
Lactate Glucose
Liver: ATP used in synthesis

of glucose (gluconeogenesis)
during recovery.
FIGURE 23–19 Metabolic cooperation between skeletal muscle and

1 ␮m
the liver: the Cori cycle. Extremely active muscles use glycogen as their
energy source, generating lactate via glycolysis. During recovery, some FIGURE 23–20 Electron micrograph of heart muscle. In the profuse mito-
of this lactate is transported to the liver and converted to glucose via chondria of heart tissue, pyruvate (from glucose), fatty acids, and ketone
gluconeogenesis. This glucose is released to the blood and returned bodies are oxidized to drive ATP synthesis. This steady aerobic metabo-
to the muscles to replenish their glycogen stores. The overall pathway lism allows the human heart to pump blood at a rate of nearly 6 L/min, or
(glucose S lactate S glucose) constitutes the Cori cycle. about 350 L/h—or 200 3 106 L of blood over 70 years.
19.2 ATP Synthesis 715
Intermembrane Malate– Matrix

OH
space -ketoglutarate

OOC CH2 C COO transporter OH

H OOC CH2 C COO
2
NAD+ H NAD+
Malate Malate
1 3
malate malate
H+ + NADH dehydrogenase dehydrogenase NADH + H+
O

OOC CH2 C COO
Oxaloacetate O
NH3 NH3 Oxaloacetate

OOC CH2 C COO
OOC CH2 CH2 C COO OOC CH2 CH2 C COO
H H
Glutamate Glutamate
aspartate aspartate
aminotransferase 6 4 aminotransferase
-Ketoglutarate -Ketoglutarate
O O

OOC CH2 CH2 C COO
OOC CH2 CH2 C COO

NH3
NH3 Aspartate Aspartate

OOC CH2 C COO
OOC CH2 C COO
H
H
5
Glutamate-aspartate
transporter
FIGURE 19–27 Malate-aspartate shuttle. This shuttle for transporting two reducing equivalents to NAD, and the resulting NADH is oxi-
reducing equivalents from cytosolic NADH into the mitochondrial ma- dized by the respiratory chain. The oxaloacetate formed from malate
trix is used in liver, kidney, and heart. 1 NADH in the cytosol (in- cannot pass directly into the cytosol. 4 It is first transaminated to as-
termembrane space) passes two reducing equivalents to oxaloacetate, partate, which 5 can leave via the glutamate-aspartate transporter.
producing malate. 2 Malate crosses the inner membrane via the 6 Oxaloacetate is regenerated in the cytosol, completing the cycle.
malate–-ketoglutarate transporter. 3 In the matrix, malate passes
Glycolysis
NAD+ cytosolic NADH + H+

glycerol 3-phosphate
dehydrogenase
CH2OH
–
C–
–O
–
FIGURE 19–28 Glycerol 3-phosphate shuttle. This alternative Glycerol 3- Dihydroxyacetone

phosphate phosphate CH2 – O – P
means of moving reducing equivalents from the cytosol to the
mitochondrial matrix operates in skeletal muscle and the CH2OH mitochondrial
–
brain. In the cytosol, dihydroxyacetone phosphate accepts CHOH glycerol 3-phosphate

dehydrogenase
–
two reducing equivalents from NADH in a reaction catalyzed

CH2 – O – P FAD
by cytosolic glycerol 3-phosphate dehydrogenase. An isozyme
FADH2
of glycerol 3-phosphate dehydrogenase bound to the outer
face of the inner membrane then transfers two reducing Q III
equivalents from glycerol 3-phosphate in the intermembrane
space to ubiquinone. Note that this shuttle does not involve
membrane transport systems. Matrix
796 Chapter 21 Lipid Biosynthesis
Inner Outer
membrane membrane
Matrix Cytosol
Citrate
transporter
Glucose CoAOSH
Citrate Citrate
CoA-SH
ATP Fatty acid
synthesis
Pyruvate
citrate ADP + Pi
pyruvate synthase
dehydrogenase citrate Acetyl-CoA
Acetyl-CoA
lyase
Amino acids Oxaloacetate Oxaloacetate

NADH + H+ NADH + H+
malate malate
dehydrogenase dehydrogenase
NAD+ NAD+
Malate Malate
ADP + Pi pyruvate
NADP+
carboxylase malic
enzyme
ATP Malate – NADPH + H+

α -ketoglutarate
CO2 CO2
transporter
Pyruvate Pyruvate
Pyruvate
transporter
FIGURE 21–10 Shuttle for transfer of acetyl groups from mitochon- livered as acetyl-CoA for fatty acid synthesis. Oxaloacetate is reduced
dria to the cytosol. The mitochondrial outer membrane is freely per- to malate, which returns to the mitochondrial matrix and is converted
meable to all these compounds. Pyruvate derived from amino acid to oxaloacetate. An alternative fate for cytosolic malate is oxidation
catabolism in the mitochondrial matrix, or from glucose by glycolysis by malic enzyme to generate cytosolic NADPH; the pyruvate pro-
in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups duced returns to the mitochondrial matrix.
pass out of the mitochondrion as citrate; in the cytosol they are de-
carboxylase is the rate-limiting step in the biosynthesis of acetyl-CoA carboxylase. At the same time, citrate in-
of fatty acids, and this enzyme is an important site of hibits the activity of phosphofructokinase-1 (see Fig.
regulation. In vertebrates, palmitoyl-CoA, the principal 15–18), reducing the flow of carbon through glycolysis.
product of fatty acid synthesis, is a feedback inhibitor Acetyl-CoA carboxylase is also regulated by cova-
of the enzyme; citrate is an allosteric activator (Fig. lent modification. Phosphorylation, triggered by the
21–11a), increasing Vmax. Citrate plays a central role in hormones glucagon and epinephrine, inactivates the
diverting cellular metabolism from the consumption enzyme and reduces its sensitivity to activation by cit-
(oxidation) of metabolic fuel to the storage of fuel as fatty rate, thereby slowing fatty acid synthesis. In its active
acids. When the concentrations of mitochondrial acetyl- (dephosphorylated) form, acetyl-CoA carboxylase poly-
CoA and ATP increase, citrate is transported out of mi- merizes into long filaments (Fig. 21–11b); phosphoryla-
tochondria; it then becomes both the precursor of cyto- tion is accompanied by dissociation into monomeric
solic acetyl-CoA and an allosteric signal for the activation subunits and loss of activity.

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58 Water

2.2 Ionization of Water, Weak Acids, H

and Weak Bases O H

tration of hydrogen ions is 1.0 3 1027 M, the pH can be

[H1] (M) pH [OH2] (M) pOH* 3 Cola, vinegar

1022 2 10212 12 5 Black cofee

Glycine, carboxyl NH⫹

Equilibrium constants for ionization reactions are usu- 9

Titration Curves Reveal the pKa of Weak Acids 0 50 100%

dissociated is the imidazole group, which can be proton-

CO2 (g) ∆ CO2 (d)

Figure 7–3 shows the structures of the D stereo-

Three carbons Four carbons Five carbons

CH2OH CH2OH CH2OH CH2OH CH2OH

acid, as in N-acetylglucosamine. This glucosamine corresponding uronic acid: glucuronic, galacturonic, or

BOX 7–1 Blood Glucose Measurements in the Diagnosis and

BOX 7–2 Sugar Is Sweet, and So Are . . . a Few Other Things

GTP-binding protein GTP-binding protein

FIGURE 2 The receptor for sweet-tasting substances, with its regions

7.2 Polysaccharides Homopolysaccharides Heteropolysaccharides

OH OH genetic studies have revealed that genes encoding

FIGURE 7–16 Chitin. (a) A short segment of chitin, a homopoly-

electrostatic interactions. Because polysaccharides O 4

atom bridging two carbon atoms (the glycosidic bond). O O 1

(a) (b) 䊉 ␾,␺ 170, 170

Keratan sulfates (Greek keras, “horn”) have no

FIGURE 7–23 Interaction between a glycosaminoglycan and its binding pro-

protein. Heparin was used in this experiment, but the glycosaminoglycan

Heparin binding causes antithrombin to bind to and

TABLE 7–2 Structures and Roles of Some Polysaccharides

Glycoproteins, and Glycosphingolipids Outside

In addition to their important roles as stored fuels

BIOENERGETICS AND METABOLISM

oxygen from water in the process. Heterotrophs in turn N2 in Denitrifying

oxidation reactions that produce carbon dioxide also

All living organisms also require a source of nitro- Nitrifying bacteria

Rubber Carotenoid Steroid

Phospholipids Isopentenyl- Bile

Mevalonate Vitamin K Cholesteryl

Sucrose Serine Leucine

(a) Converging catabolism

(b) Payof phase O

ﬁrst ATP-forming reaction 2ADP 7

second ATP-forming reaction 2ADP P

1 2 3 4 5 6 Phosphorylation molecule; the two molecules of pyruvate formed by

Glucose responsible for the stimulation. Harden and Young soon

This reaction, which is irreversible under intracellular con-

The different hexokinase isozymes of liver and

Relative enzyme activity

gluconeogenic enzyme that bypasses the hexokinase step Allosteric

tion, act allosterically to relieve this inhibition by ATP.

Gluconeogenesis needs. One source of glucose is glycogen stored in the

Fructose 2,6-Bisphosphate Is a Potent Allosteric OH H

ATP Fructose 6-phosphate Pi

PFK-1 F26BP FBPase-1

ADP Fructose 1,6-bisphosphate H2O

Fructose 6-phosphate Pi ATP

Liver only All glycolytic tissues, including liver

ADP ATP 6 steps