ch03 10/11/01 11:08 PM Page 57

Enzymes

3.1. INTRODUCTION

Enzymes are usually proteins of high molecular weight (15,000 < MW < several million
daltons) that act as catalysts. Recently, it has been shown that some RNA molecules are
also catalytic, but the vast majority of cellular reactions are mediated by protein catalysts.
RNA molecules that have catalytic properties are called ribozymes. Enzymes are specific,
versatile, and very effective biological catalysts, resulting in much higher reaction rates as
compared to chemically catalyzed reactions under ambient conditions. More than 2000
enzymes are known. Enzymes are named by adding the suffix -ase to the end of the sub-
strate, such as urease, or the reaction catalyzed, such as alcohol dehydrogenase. Some en-
zymes have a simple structure, such as a folded polypeptide chain (typical of most
hydrolytic enzymes). Many enzymes have more than one subunit. Some protein enzymes
require a nonprotein group for their activity. This group is either a cofactor, such as metal
ions, Mg, Zn, Mn, Fe, or a coenzyme, such as a complex organic molecule, NAD, FAD,
CoA, or some vitamins. An enzyme containing a nonprotein group is called a holoenzyme.
The protein part of this enzyme is the apoenzyme (holoenzyme = apoenzyme +
cofactor). Enzymes that occur in several different molecular forms, but catalyze the same
reaction, are called isozymes. Some enzymes are grouped together to form enzyme com-
plexes. Enzymes are substrate specific and are classified according to the reaction they
catalyze. Major classes of enzymes and their functions are listed in Table 3.1.

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TABLE 3.1 International Classification of Enzymes: Class Names, Code Numbers, and Types
of Reactions Catalyzed

1. Oxidoreductases (oxidation- 3. Hydrolases (hydrolysis reactions)

reduction reactions) 3.1 Esters
3.2 Glycosidic bonds
1.1 Acting on 3.4 Peptide bonds
3.5 Other C—N bonds
3.6 Acid anhydrides
4. Lyases (addition to double bonds)
1.2 Acting on

4.1

1.3 Acting on

4.2

1.4 Acting on

4.3

1.5 Acting on

1.6 Acting on NADH; NADPH

2. Transferases (transfer of 5. Isomerases (isomerization reactions)
functional groups) 5.1 Racemases
2.1 One-carbon groups 6. Ligases (formation of bonds with
2.2 Aldehydic or ketonic groups ATP cleavage)
2.3 Acyl groups 6.1 C—O
2.4 Glycosyl groups 6.2 C—S
2.7 Phosphate groups 6.3 C—N
2.8 S-containing groups 6.4 C—C

With permission, from A. Lehninger, Biochemistry, 2d edition, Worth Publishers, New York, 1975.

3.2. HOW ENZYMES WORK

Enzymes lower the activation energy of the reaction catalyzed by binding the substrate
and forming an enzyme–substrate complex. Enzymes do not affect the free-energy change
or the equilibrium constant. Figure 3.1 illustrates the action of an enzyme from the
activation-energy point of view. For example, the activation energy for the decomposition
of hydrogen peroxide varies depending on the type of catalysis. The activation energy of
the uncatalyzed reaction at 20∞C is 18 kilocalories per mole (kcal/mol), whereas the DE
values for chemically catalyzed (by colloidal platinum) and enzymatically catalyzed
(catalase) decomposition are 13 and 7 kcal/mol, respectively. That is, catalase accelerates
the rate of reaction by a factor of about 108. The reader should note that this large change
in rate for a relatively small change in activation energy is due to the exponential depen-
dence of rate on activation energy. In this case, the ratio of the rates is exp(-7000/2 ◊ 293)
∏ exp(-18,000/2 ◊ 293).

58 Enzymes Chap. 3
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Figure 3.1. Activation energies of enzy-

matically catalyzed and uncatalyzed reac-
tions. Note that |DG°A2|<|DG°A1|.

The molecular aspects of enzyme–substrate interaction are not yet fully understood.
This interaction varies from one enzyme–substrate complex to another. Various studies
using x-ray and Raman spectroscopy have revealed the presence of the enzyme–substrate
(ES) complex. The interaction between the enzyme and its substrate is usually by weak
forces. In most cases, van der Waals forces and hydrogen bonding are responsible for the
formation of ES complexes. The substrate binds to a specific site on the enzyme known as
the active site. The substrate is a relatively small molecule and fits into a certain region on
the enzyme molecule, which is a much larger molecule. The simplest model describing
this interaction is the lock-and-key model, in which the enzyme represents the lock and
the substrate represents the key, as described in Fig. 3.2.
In multisubstrate enzyme-catalyzed reactions, enzymes can hold substrates such
that reactive regions of substrates are close to each other and to the enzyme’s active site,
which is known as the proximity effect. Also, enzymes may hold the substrates at certain
positions and angles to improve the reaction rate, which is known as the orientation effect.
In some enzymes, the formation of an enzyme–substrate complex causes slight changes in
the three-dimensional shape of the enzyme. This induced fit of the substrate to the enzyme
molecule may contribute to the catalytic activity of the enzyme, too. The enzymes
lysozyme and carboxypeptidase A have been observed to change their three-dimensional
structure upon complexing with the substrate. Enzyme catalysis is affected not only by the
primary structure of enzymes but also by the secondary, tertiary, and quaternary struc-
tures. The properties of the active site of enzymes and the folding characteristics have a
profound effect on the catalytic activity of enzymes. Certain enzymes require coenzymes
and cofactors for proper functioning. Table 3.2 lists some enzymes and their cofactors and
coenzymes.

Figure 3.2. Schematic of the lock-and-key

model of enzyme catalysis.

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TABLE 3.2 Cofactors (Metal Ions) and Coenzymes of Some Enzymes

Coenzyme Entity transferred

Zn2+ Nicotinamide adenine dinucleotide Hydrogen atoms (electrons)
Alcohol dehydrogenase Nicotinamide adenine dinucleotide Hydrogen atoms (electrons)
Carbonic anhydrase phosphate Hydrogen atoms (electrons)
Carboxypeptidase Flavin mononucleotide Hydrogen atoms (electrons)
Mg2+ Flavin adenine dinucleotide Hydrogen atoms (electrons)
Phosphohydrolases Coenzyme Q Aldehydes
Phosphotransferases Thiamin pyrophosphate Acyl groups
Mn2+ Coenzyme A Acyl groups
Arginase Lipoamide Alkyl groups
Phosphotransferases Cobamide coenzymes Carbon dioxide
Fe2+ or Fe3+ Biocytin Amino groups
Cytochromes Pyridoxal phosphate Methyl, methylene, formyl,
Peroxidase Tetrahydrofolate coenzymes or formimino groups
Catalase
Ferredoxin
Cu2+ (Cu+)
Tyrosinase
Cytochrome oxidase
K+
Pyruvate kinase (also requires Mg2+)
Na+
Plasma membrane ATPase (also
requires K+ and Mg2+)

With permission, from A. Lehninger, Biochemistry, 2d ed., Worth Publishers, New York, 1975.

3.3. ENZYME KINETICS

3.3.1. Introduction

A mathematical model of the kinetics of single-substrate-enzyme-catalyzed reactions was

first developed by V. C. R. Henri in 1902 and by L. Michaelis and M. L. Menten in 1913.
Kinetics of simple enzyme-catalyzed reactions are often referred to as Michaelis–Menten
kinetics or saturation kinetics. The qualitative features of enzyme kinetics are similar to
Langmuir–Hinshelwood kinetics (see Fig. 3.3). These models are based on data from
batch reactors with constant liquid volume in which the initial substrate, [S0], and en-
zyme, [E0], concentrations are known. More complicated enzyme-substrate interactions
such as multisubstrate–multienzyme reactions can take place in biological systems. An
enzyme solution has a fixed number of active sites to which substrates can bind. At high
substrate concentrations, all these sites may be occupied by substrates or the enzyme is
saturated. Saturation kinetics can be obtained from a simple reaction scheme that involves
a reversible step for enzyme–substrate complex formation and a dissociation step of the
ES complex.
k1 k2
E + S a ES ææÆ E + P (3.1)
k-1

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Figure 3.3. Effect of substrate concentra-

tion on the rate of an enzyme-catalyzed re-
action.

It is assumed that the ES complex is established rather rapidly and the rate of the re-
verse reaction of the second step is negligible. The assumption of an irreversible second
reaction often holds only when product accumulation is negligible at the beginning of the
reaction. Two major approaches used in developing a rate expression for the enzyme-
catalyzed reactions are (1) rapid-equilibrium approach and (2) quasi-steady-state approach.

3.3.2. Mechanistic Models for Simple Enzyme Kinetics

Both the quasi-steady-state approximation and the assumption of rapid equilibrium share
the same few initial steps in deriving a rate expression for the mechanism in eq. 3.1,
where the rate of product formation is
d[ P ]
v= = k2 [ ES] (3.2)
dt
where v is the rate of product formation or substrate consumption in moles/l-s.
The rate constant k2 is often denoted as kcat in the biological literature. The rate of
variation of the ES complex is
d[ ES]
= k1[ E ][S] - k-1[ ES] - k2 [ ES] (3.3)
dt
Since the enzyme is not consumed, the conservation equation on the enzyme yields
[ E ] = [ E 0 ] - [ ES] (3.4)

At this point, an assumption is required to achieve an analytical solution.

3.3.2.1.The rapid equilibrium assumption. Henri and Michaelis and Menten

used essentially this approach. Assuming a rapid equilibrium between the enzyme and

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substrate to form an [ES] complex, we can use the equilibrium coefficient to express [ES] in
terms of [S].
The equilibrium constant is
k [ E ][S]
K m = -1 = (3.5)
k1 [ ES]
Since [E] = [E0] - [ES] if enzyme is conserved, then
[ E 0 ][S]
[ ES] = (3.6)
( k-1 / k1 ) + [S]

[ E 0 ][S] (3.7)
[ ES] =
Km + [S]

where Km¢ = k-1/k1, which is the dissociation constant of the ES complex. Substituting eq.
3.7 into eq. 3.2 yields
d[ P ] [ E 0 ][S] Vm [S]
v= = k2 = (3.8)
dt Km + [S] Km + [S]
where Vm = k2[E0].
In this case, the maximum forward velocity of the reaction is Vm. Vm changes if
more enzyme is added, but the addition of more substrate has no influence on Vm. K¢m is
often called the Michaelis–Menten constant, and the prime reminds us that it was derived
by assuming rapid equilibrium in the first step. A low value of K¢m suggests that the en-
zyme has a high affinity for the substrate. Also, K¢m corresponds to the substrate concentra-
tion, giving the half-maximal reaction velocity.
An equation of exactly the same form as eq. 3.8 can be derived with a different,
more general assumption applied to the reaction scheme in eq. 3.1.

3.3.2.2. The quasi-steady-state assumption. In many cases the assumption

of rapid equilibrium following mass-action kinetics is not valid, although the enzyme–
substrate reaction still shows saturation-type kinetics.
G. E. Briggs and J. B. S. Haldane first proposed using the quasi-steady-state as-
sumption. In most experimental systems a closed system (batch reactor) is used in which
the initial substrate concentration greatly exceeds the initial enzyme concentration. They
suggest that since [E0] was small, d[ES]/dt ª 0. (This logic is flawed. Do you see why?)
Computer simulations of the actual time course represented by eqs. 3.2, 3.3, and 3.4 have
shown that in a closed system the quasi-steady-state hypothesis holds after a brief tran-
sient if [S0]  [E0] (for example, 100¥). Figure 3.4 displays one such time course.
By applying the quasi-steady-state assumption to eq. 3.3, we find
k [ E ][S]
[ ES] = 1 (3.9)
k-1 + k2

Substituting the enzyme conservation eq. 3.4 in eq. 3.9 yields

k ([ E ] - [ ES])[S]
[ ES] = 1 0 (3.10)
k-1 + k2

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Figure 3.4. Time course of the formation

of an enzyme/substrate complex and initia-
tion of the steady state, as derived from
computer solutions of data obtained in an
actual experiment on a typical enzyme. The
portion in the dashed box in the top graph is
shown in magnified form on the lower
graph. (With permission, adapted from A.
Lehninger, Biochemistry, 2d ed., Worth Pub-
lishers, New York, 1975, p. 191.)

Solving eq. 3.10 for [ES],

[ E 0 ][S]
[ ES] =
k-1 + k2 (3.11)
+ [S]
k1

Substituting eq. 3.11 into eq. 3.2 yields

d[ P ] k2 [ E 0 ][S]
v= =
dt k -1 + k2 (3.12a)
+ [S]
k1

Vm [S]
v= (3.12b)
K m + [S]

where Km is (k-1 + k2)/k1 and Vm is k2[E0]. Under most circumstances (simple experi-
ments), it is impossible to determine whether Km or K¢m is more suitable. Since Km results
from the more general derivation, we will use it in the rest of our discussions.

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3.3.3. Experimentally Determining Rate Parameters

for Michaelis–Menten Type Kinetics

The determination of values for Km and Vm with high precision can be difficult. Typically,
experimental data are obtained from initial-rate experiments. A batch reactor is charged
with a known amount of substrate [S0] and enzyme [E0]. The product (or substrate
concentration) is plotted against time. The initial slope of this curve is estimated (i.e.,
v = d[P]/dt|t=0 = -d[S]/dt|t=0). This value of v then depends on the values of [E0] and [S0] in
the charge to the reactor. Many such experiments can be used to generate many pairs of
v and [S] data. These could be plotted as in Fig. 3.3, but the accurate determination of Km
from such a plot is very difficult. Consequently, other methods of analyzing such data
have been suggested.

3.3.3.1. Double-reciprocal plot (Lineweaver–Burk plot). Equation 3.12b

can be linearized in double-reciprocal form:

1 1 Km 1
= + (3.13)
v Vm Vm [S]

A plot of 1/v versus 1/[S] yields a linear line with a slope of Km/Vm and y-axis intercept of
1/Vm, as depicted in Fig. 3.5. A double-reciprocal plot gives good estimates on Vm, but not
necessarily on Km. Because the error about the reciprocal of a data point is not symmetric,
the reader should be cautious in applying regression analysis (least squares) to such plots.
Data points at low substrate concentrations influence the slope and intercept more than
those at high substrate concentrations.

Figure 3.5. Double-reciprocal (Lineweaver–Burk) plot.

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3.3.3.2. Eadie–Hofstee plot. Equation 3.12b can be rearranged as

v
v = Vm - K m (3.14)
[S]
A plot of v versus v/[S] results in a line of slope -Km and y-axis intercept of Vm, as de-
picted in Fig. 3.6. Eadie–Hofstee plots can be subject to large errors since both coordi-
nates contain u, but there is less bias on points at low [S].

3.3.3.3. Hanes–Woolf plot. Rearrangement of eq. 3.12b yields

[S] K m 1
= + [S] (3.15)
v Vm Vm

A plot of [S]/v versus [S] results in a line of slope 1/Vm and y-axis intercept of Km/Vm, as
depicted in Fig. 3.7. This plot is used to determine Vm more accurately.

3.3.3.4. Batch kinetics. The time course of variation of [S] in a batch enzy-
matic reaction can be determined from
d[S] V [S]
v=- = m (3.12b)
dt K m + [S]
by integration to yield
[S 0 ]
Vm t = [S0 ] - [S] + K m ln (3.16)
[S]

Figure 3.6. Eadie–Hofstee plot.

Sec. 3.3 Enzyme Kinetics 65

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Figure 3.7. Hanes–Woolf plot.

or

[S0 ] - [S] K m [S0 ]

Vm - = ln (3.17)
t t [S]

A plot of 1/t ln[S0]/[S] versus {[S0] - [S]}/t results in a line of slope -1/Km and intercept
of Vm /Km.

3.3.3.5. Interpretation of Km and Vm. While Km (or Km¢) is an intrinsic pa-

rameter, Vm is not. Km is solely a function of rate parameters and is expected to change
with temperature or pH. However, Vm is a function of the rate parameter k2 and the initial
enzyme level, [E0]. As [E0] changes, so does Vm. Of course, k2 can be readily calculated if
[E0] is known. For highly purified enzyme preparations it may be possible to express [E0]
in terms of mol/l or g/l.
When the enzyme is part of a crude preparation, its concentration is in terms of
“units.” A “unit” is the amount of enzyme that gives a predetermined amount of catalytic
activity under specific conditions. For example, one unit would be formation of one mmol
product per minute at a specified pH and temperature with a substrate concentration much
greater than the value of Km. The specific activity is the number of units of activity per
amount of total protein. For example, a crude cell lysate might have a specific activity of
0.2 units/mg protein which upon purification may increase to 10 units/mg protein. Only
enzyme that remains catalytically active will be measured. The enzyme may be denatured
if it unfolds or has its three-dimensional shape altered by pH extremes or temperature dur-
ing purification. The denatured enzyme will have no activity.

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Example 3.1.
To measure the amount of glucoamylase in a crude enzyme preparation, 1 ml of the crude en-
zyme preparation containing 8 mg protein is added to 9 ml of a 4.44% starch solution. One
unit of activity of glucoamylase is defined as the amount of enzyme which produces 1 mmol
of glucose per min in a 4% solution of Lintner starch at pH 4.5 and at 60∞C. Initial rate exper-
iments show that the reaction produces 0.6 mmol of glucose/ml-min. What is the specific ac-
tivity of the crude enzyme preparation?
Solution The total amount of glucose made is 10 ml ¥ 0.6 mmol glucose/ml-min or 6 mmol
glucose per min. The specific activity is then:
6 units
specific activity =
1 ml protein solution ◊ 8 mg/ ml
= 6 units/8 mg protein
= 0.75 units/ mg protein

Vm must have units such as mmol product/ml-min. Since Vm = k2E0, the dimensions
of k2 must reflect the definition of units in E0. In the above example we had a concentra-
tion of enzyme of 8 mg protein/10 ml solution ◊ 0.75 units/mg protein or 0.6 units/ml. If,
for example, Vm = 1 mmol/ml-min, then k2 = 1 mmol/ml-min ∏ 0.6 units/ml or k2 = 1.67
mmol/unit-min.

3.3.4. Models for More Complex Enzyme Kinetics

3.3.4.1. Allosteric enzymes. Some enzymes have more than one substrate
binding site. The binding of one substrate to the enzyme facilitates binding of other sub-
strate molecules. This behavior is known as allostery or cooperative binding, and regula-
tory enzymes show this behavior. The rate expression in this case is
n
d[S] Vm [S]
v=- = n (3.18)
dt K m≤ + [S]

where n = cooperativity coefficient and n > 1 indicates positive cooperativity. Figure 3.8
compares Michaelis–Menten kinetics with allosteric enzyme kinetics, indicating a sig-
moidal shape of u -[S] plot for allosteric enzymes.
The cooperativity coefficient can be determined by rearranging eq. 3.18 as
v
ln = n ln[S] - ln K m≤ (3.19)
Vm - v

and by plotting ln v/(Vm - v) versus ln[S] as depicted in Fig. 3.9.

3.3.4.2. Inhibited enzyme kinetics. Certain compounds may bind to en-

zymes and reduce their activity. These compounds are known to be enzyme inhibitors. En-
zyme inhibitions may be irreversible or reversible. Irreversible inhibitors such as heavy
metals (lead, cadium, mercury, and others) form a stable complex with enzyme and re-
duce enzyme activity. Such enzyme inhibition may be reversed only by using chelating
agents such as EDTA (ethylenediaminetetraacetic acid) and citrate. Reversible inhibitors
may dissociate more easily from the enzyme after binding. The three major classes of re-

Sec. 3.3 Enzyme Kinetics 67

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Figure 3.8. Comparison of Michaelis–

Menten and allosteric enzyme kinetics.

versible enzyme inhibitions are competitive, noncompetitive, and uncompetitive inhibi-

tions. The substrate may act as an inhibitor in some cases.
Competitive inhibitors are usually substrate analogs and compete with substrate for the
active site of the enzyme. The competitive enzyme inhibition scheme can be described as
E+S aES ææÆ E + P
k1
k-1
k2

+
I
a

K1 (3.20)

EI

Figure 3.9. Determination of cooperativity

coefficient.

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Assuming rapid equilibrium and with the definition of

[ E ][S] [ E ][I]
K m¢ = , KI =
[ ES] [ EI] (3.21)
[ E 0 ] = [ E ] + [ ES] + [ EI] and v = k2 [ ES]

we can develop the following equation for the rate of enzymatic conversion:
Vm [S]
v=
È [I] ˘ (3.22)
K m¢ Í1 + ˙ + [S]
Î KI˚

or
Vm [S]
v= (3.23)
K m¢ , app + [S]

  Ê [I] ˆ
where K m,app = K m Á1 + ˜
Ë K1 ¯
.
The net effect of competitive inhibition is an increased value of K¢m , app and, there-
fore, reduced reaction rate. Competitive inhibition can be overcome by high concentra-
tions of substrate. Figure 3.10 describes competitive enzyme inhibition in the form of a
double-reciprocal plot.
Noncompetitive inhibitors are not substrate analogs. Inhibitors bind on sites other
than the active site and reduce enzyme affinity to the substrate. Noncompetitive enzyme
inhibition can be described as follows:

E+S aES ææÆ E + P

K ¢m
k2

+ +
I I
(3.24)
a
a

KI
EI + S aESI K ¢m

With the definition of

[ E ][S] [ EI][S] [ E ][I] [ ES][I]
K m = = , KI = =
[ ES] [ ESI] [ EI] [ ESI] (3.25)
[ E 0 ] = [E] + [ES] + [EI] + [ESI] and v = k2 [ ES]

we can develop the following rate equation:

Vm
v=
Ê [I] ˆ Ê K m¢ ˆ (3.26)
Á1 + ˜ ÁË1 + ˜
Ë K1 ¯ [S] ¯

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Figure 3.10. Different forms of inhibited enzyme kinetics.

or

Vm, app
v=
Ê K m¢ ˆ (3.27)
Á1 + ˜
Ë [S] ¯

Vm
where Vm, app =
Ê [I] ˆ
Á1 + ˜
Ë K1 ¯

The net effect of noncompetitive inhibition is a reduction in Vm. High substrate con-
centrations would not overcome noncompetitive inhibition. Other reagents need to be
added to block binding of the inhibitor to the enzyme. In some forms of noncompetitive
inhibition Vm is reduced and K¢m is increased. This occurs if the complex ESI can form
product.
Uncompetitive inhibitors bind to the ES complex only and have no affinity for the
enzyme itself. The scheme for uncompetitive inhibition is

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K m¢
E+S aES ææÆ E + P k2

+
I

a
(3.28)
K1

ESI

With the definition of

[ E ][S] [ ES][I]
K m¢ = , K1 =
[ ES] [ ESI] (3.29)
[ E 0 ] = [ E ] + [ ES] + [ ESI] and v = k2 [ ES]

we can develop the following equation for the rate of reaction:

Vm
[S]
Ê [I] ˆ
Á 1 + ˜
Ë Kl ¯
v=
K m¢ (3.30)
+ [S]
Ê [I] ˆ
Á1 + ˜
Ë Kl ¯

or
Vm,app [S]
v= (3.31)
K m¢ , app + [S]

The net effect of uncompetitive inhibition is a reduction in both Vm and K¢m values.
Reduction in Vm has a more pronounced effect than the reduction in K¢m , and the net result
is a reduction in reaction rate. Uncompetitive inhibition is described in Fig. 3.10 in the
form of a double-reciprocal plot.
High substrate concentrations may cause inhibition in some enzymatic reactions,
known as substrate inhibition. Substrate inhibition is graphically described in Fig. 3.11.
The reaction scheme for uncompetitive substrate inhibition is

Figure 3.11. Comparison of substrate-

inhibited and uninhibited enzymatic
reactions.

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K m¢
E+S aES ææÆ E + P k2

+
S

a
KS1 (3.32)

ES2

With the definitions of

K S1 =
[S][ES] , K m¢ =
[S][E] (3.33)
[ ES2 ] [ES]
the assumption of rapid equilibrium yields
Vm [S]
v=
[S]2 (3.34)
K m¢ + [S] +
KS1

A double-reciprocal plot describing substrate inhibition is given in Fig. 3.10.

At low substrate concentrations, [S]2/KS << 1, and inhibition effect is not observed.
1
The rate is
Vm
v=
È K m¢ ˘ (3.35)
Í1 + ˙
Î [ ]˚
S

or
1 1 K m¢ 1
= + (3.36)
v Vm Vm [S]
A plot of 1/v versus 1/[S] results in a line of slope K¢m/Vm and intercept of 1/Vm.
At high substrate concentrations, K¢m/[S] << 1, and inhibition is dominant. The rate
in this case is
Vm
v=
Ê [S] ˆ (3.37)
Á1 + K ˜
Ë S1 ¯

or

1 1
= +
[S]
(3.38)
v Vm K S1 Vm

A plot of 1/v versus [S] results in a line of slope 1/Ks1 ◊ Vm and intercept of 1/Vm.

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The substrate concentration resulting in the maximum reaction rate can be determined by
setting dv/d[S] = 0. The [S]max is given by

[S]max = K m¢ KS1 (3.39)

Example 3.2
The following data have been obtained for two different initial enzyme concentrations for an
enzyme-catalyzed reaction.

v([E0] = 0.015 g/l) [S] v([E0] = 0.00875 g/l)

(g/l-min) (g/l) (g/l-min)

1.14 20.0 0.67

0.87 10.0 0.51
0.70 6.7 0.41
0.59 5.0 0.34
0.50 4.0 0.29
0.44 3.3
0.39 2.9
0.35 2.5

a. Find Km.
b. Find Vm for [E0] = 0.015 g/l.
c. Find Vm for [E0] = 0.00875 g/l.
d. Find k2.
Solution A Hanes–Woolf plot (Fig. 3.12) can be used to determine Vm and Km.

[S] Km 1
= + [S]
v Vm Vm

[S]/v (E0 = 0.015) [S]/ v (E0 = 0.00875) [S]

(min) (min) (g/l)

17.5 30 20.0
11.5 20 10.0
9.6 16 6.7
8.5 15 5.0
8.0 14 4.0
7.6 3.3
7.3 2.9
7.1 2.5

From a plot of [S]/v versus [S] for E0 = 0.015 g/l, the slope is found to be
0.6 min/g/l and Vm = 1/0.6 = 1.7 g/l min. The y-axis intercept is Km/Vm = 5.5 min and Km = 9.2
g [S]/l.
Also, Vm = k2E0 and k2 = 1.7/0.015 = 110 g/g enzyme-min. The Hanes–Woolf plot for
E0 = 0.00875 g/l gives a slope of 1.0 min/g/l and Vm = 1.0 g/l-min; k2 = Vm/E0 = 1.0/0.00875 =
114 g/g enzyme-min.

Sec. 3.3 Enzyme Kinetics 73

ch03 10/11/01 11:08 PM Page 74

Figure 3.12. Hanes–Woolf plots for E0 = 0.015 g/l and E0 = 0.00875 g/l (Example 3.1).

Example 3.3
The hydrolysis of urea by urease is an only partially understood reaction and shows inhibi-
tion. Data for the hydrolysis of the reaction are given next.

Substrate concentration: 0.2 M 0.02 M

l/v I l/v I

0.22 0 0.68 0
0.33 0.0012 1.02 0.0012
0.51 0.0027 1.50 0.0022
0.76 0.0044 1.83 0.0032
0.88 0.0061 2.04 0.0037
1.10 0.0080 2.72 0.0044
1.15 0.0093 3.46 0.0059

where v = moles/l-min and I is inhibitor molar concentration.

a. Determine the Michaelis–Menten constant (K¢m) for this reaction.
b. What type of inhibition reaction is this? Substantiate the answer.
c. Based on the answer to part b, what is the value of Ki?
Solution A double-reciprocal plot of l/v versus l/[S] for inhibitor concentrations I = 0,
0.0012, 0.0044, and 0.006 indicates that the inhibition is noncompetitive (Fig. 3.13). From
the x-axis intercept of the plot, -1/Km¢ = -13 and Km¢ ª 7.77 ¥ 10-2 M. For [S] = 0.2 M and
I = 0 from the intercept of 1/V versus 1/S, l/vm = 0.2 and Vm @ 5 moles/l-min. For I = 0.0012
M and [S] = 0.2 M, v = 3 moles/l-min. Substituting these values in
Vm
v=
Ê [I] ˆ Ê Km¢ ˆ
Á1 + K ˜ ÁË1 + [S] ˜¯
Ë I¯

gives KI = 6 ¥ 10-3 M.

74 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 75

Figure 3.13. Double-reciprocal plot for different inhibitor concentrations (Example 3.2).

3.3.5. Effects of pH and Temperature

3.3.5.1. pH effects. Certain enzymes have ionic groups on their active sites,
and these ionic groups must be in a suitable form (acid or base) to function. Variations in
the pH of the medium result in changes in the ionic form of the active site and changes in
the activity of the enzyme and hence the reaction rate. Changes in pH may also alter the
three-dimensional shape of the enzyme. For these reasons, enzymes are only active over a
certain pH range. The pH of the medium may affect the maximum reaction rate, Km, and
the stability of the enzyme. In some cases, the substrate may contain ionic groups, and the
pH of the medium affects the affinity of the substrate to the enzyme.
The following scheme may be used to describe pH dependence of the enzymatic re-
action rate for ionizing enzymes.
- +
E +H
a

K2
Km¢ k2
EH + S
+
aEHS ææÆ EH + P
+
H
a

K1 (3.40)
+
EH 2

With the definition of

Sec. 3.3 Enzyme Kinetics 75

ch03 10/11/01 11:08 PM Page 76

[ EH][S]
K m¢ =
[ EHS]

[ EH][H + ]
K1 =
[ EH +2 ] (3.41)

[ E - ][H + ]
K2 =
[ EH]
[E 0 ] = [E - ] + [ EH] + [ EH +2 ] + [ EHS], v = k2 [ EHS]

We can derive the following rate expression:

Vm [S]
v= +
È K [H ] ˘
K m¢ Í1 + 2+ + + [S] (3.42)
Î [H ] K1 ˙˚

or
Vm [S]
v= (3.43)
K ¢ + [S]
m ,app

È K [H + ] ˘
where K m,app = K m Í1 + +2 + ˙
Î [H ] K1 ˚
As a result of this behavior, the pH optimum of the enzyme is between pK1 and pK2.
For the case of ionizing substrate, the following scheme and rate expression can be
developed:
Km¢
SH + E
+
aESH + k2
ææ Æ E + HP
+
a

K1 (3.44)
+
S+H

Vm [S]
v=
Ê K ˆ (3.45)
K m¢ Á1 + +1 ˜ + [S]
Ë [H ] ¯

Theoretical prediction of the pH optimum of enzymes requires a knowledge of the

active site characteristics of enzymes, which are very difficult to obtain. The pH optimum
for an enzyme is usually determined experimentally. Figure 3.14 depicts variation of en-
zymatic activity with pH for two different enzymes.

3.3.5.2. Temperature effects. The rate of enzyme-catalyzed reactions in-

creases with temperature up to a certain limit. Above a certain temperature, enzyme activ-

76 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 77

Figure 3.14. The pH-activity profiles of

two enzymes. (A) approximate activity for
trypsin; (B) approximate activity for
cholinesterase.

ity decreases with temperature because of enzyme denaturation. Figure 3.15 depicts the
variation of reaction rate with temperature and the presence of an optimal temperature.
The ascending part of Fig. 3.15 is known as temperature activation. The rate varies ac-
cording to the Arrhenius equation in this region.
v = k2 [ E ] (3.46a)

k2 = Ae - Ea RT (3.46b)

Figure 3.15. Effect of temperature on the

activity of an enzyme. Here we have as-
sumed a value of Ea = 11 kcal/g-mol and
Ed = 70 kcal/g-mol. The descending portion
of the curve is due to thermal denaturation
and is calculated assuming a 10-min expo-
sure to the temperature. Note that the nature
of the plot will depend on the length of time
the reaction mixture is exposed to the test
temperature.

Sec. 3.3 Enzyme Kinetics 77

ch03 10/11/01 11:08 PM Page 78

where Ea is the activation energy (kcal/mol) and [E] is the active enzyme concentration. A
plot of ln u versus 1/T results in a line of slope -Ea/R.
The descending part of Fig. 3.15 is known as temperature inactivation or thermal
denaturation. The kinetics of thermal denaturation can be expressed as
d[ E ]
- = kd [ E ] (3.47)
dt
or

[ E ] = [ E 0 ]e - kd t (3.48)

where [E0] is the initial enzyme concentration and kd is the denaturation constant. kd also
varies with temperature according to the Arrhenius equation.

kd = Ad e - Ea RT
(3.49)

where Ed is the deactivation energy (kcal/mol). Consequently,

v = Ae - Ea RT
E 0 e - kd t (3.50)

The activation energies of enzyme-catalyzed reactions are within the 4 to 20 kcal/g

mol range (mostly about 11 kcal/g mol). Deactivation energies Ed vary between 40 and
130 kcal/g mol (mostly about 70 kcal/g mol). That is, enzyme denaturation by tempera-
ture is much faster than enzyme activation. A rise in temperature from 30∞ to 40∞C results
in a 1.8-fold increase in enzyme activity, but a 41-fold increase in enzyme denaturation.
Variations in temperature may affect both Vm and Km values of enzymes.

3.3.6. Insoluble Substrates

Enzymes are often used to attack large, insoluble substrates such as wood chips (in bio-
pulping for paper manufacture) or cellulosic residues from agriculture (e.g., cornstalks).
In these cases access to the reaction site on these biopolymers by enzymes is often limited
by enzyme diffusion. The number of potential reactive sites exceeds the number of en-
zyme molecules. This situation is opposite that of the typical situation with soluble sub-
strates, where access to the enzyme’s active site limits reaction. If we consider initial
reaction rates and if the reaction is first order with respect to the concentration of enzyme
bound to substrate (i.e., [ES]), then we can derive a rate expression:

Vmax,S [ E ]
v= (3.51a)
K eq + [ E ]

where
Vmax,S = k2 [S0] (3.51b)

and
¢ = k /k
Keq (3.51c)
des ads

78 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 79

The previous equation assumes slow binding of enzyme (i.e., [E] ª [E0]), S0 is the number
of substrate bonds available initially for breakage, and kdes and kads refer to rates of en-
zyme desorption and adsorption onto the insoluble matrix, respectively.

3.4 IMMOBILIZED ENZYME SYSTEMS

The restriction of enzyme mobility in a fixed space is known as enzyme immobilization.

Immobilization of enzymes provides important advantages, such as enzyme reutilization
and elimination of enzyme recovery and purification processes, and may provide a better
environment for enzyme activity. Since enzymes are expensive, catalyst reuse is critical
for many processes. Since some of the intracellular enzymes are membrane bound, immo-
bilized enzymes provide a model system to mimic and understand the action of some
membrane-bound intracellular enzymes. Product purity is usually improved, and effluent
handling problems are minimized by immobilization.

3.4.1. Methods of Immobilization

Major methods of immobilization are summarized in Fig. 3.16. The two major categories
are entrapment and surface immobilization.

3.4.1.1. Entrapment. Entrapment is the physical enclosure of enzymes in a

small space. Matrix entrapment and membrane entrapment, including microencapsula-
tion, are the two major methods of entrapment.
Matrices used for enzyme immobilization are usually polymeric materials such as
Ca-alginate, agar, k-carrageenin, polyacrylamide, and collagen. However, some solid ma-
trices such as activated carbon, porous ceramic, and diatomaceous earth can also be used
for this purpose. The matrix can be a particle, a membrane, or a fiber. When immobilizing

Figure 3.16. Major immobilization methods.

Sec. 3.4 Immobilized Enzyme Systems 79

ch03 10/11/01 11:08 PM Page 80

in a polymer matrix, enzyme solution is mixed with polymer solution before polymeriza-
tion takes place. Polymerized gel-containing enzyme is either extruded or a template is
used to shape the particles from a liquid polymer-enzyme mixture. Entrapment and sur-
face attachment may be used in combination in some cases.
Membrane entrapment of enzymes is possible; for example, hollow fiber units have
been used to entrap an enzyme solution between thin, semipermeable membranes. Mem-
branes of nylon, cellulose, polysulfone, and polyacrylate are commonly used. Configura-
tions, other than hollow fibers, are possible, but in all cases a semipermeable membrane is
used to retain high-molecular-weight compounds (enzyme), while allowing small-
molecular-weight compounds (substrate or products) access to the enzyme.
A special form of membrane entrapment is microencapsulation. In this technique,
microscopic hollow spheres are formed. The spheres contain the enzyme solution, while
the sphere is enclosed within a porous membrane. The membrane can be polymeric or an
enriched interfacial phase formed around a microdrop.
Despite the aforementioned advantages, enzyme entrapment may have its inherent
problems, such as enzyme leakage into solution, significant diffusional limitations, reduced
enzyme activity and stability, and lack of control of microenvironmental conditions. En-
zyme leakage can be overcome by reducing the MW cutoff of membranes or the pore size of
solid matrices. Diffusion limitations can be eliminated by reducing the particle size of ma-
trices and/or capsules. Reduced enzyme activity and stability are due to unfavorable mi-
croenvironmental conditions, which are difficult to control. However, by using different
matrices and chemical ingredients, by changing processing conditions, and by reducing par-
ticle or capsule size, more favorable microenvironmental conditions can be obtained. Diffu-
sion barrier is usually less significant in microcapsules as compared to gel beads.

3.4.1.2. Surface immobilization. The two major types of immobilization of

enzymes on the surfaces of support materials are adsorption and covalent binding.
Adsorption is the attachment of enzymes on the surfaces of support particles by weak
physical forces, such as van der Waals or dispersion forces. The active site of the adsorbed
enzyme is usually unaffected, and nearly full activity is retained upon adsorption. However,
desorption of enzymes is a common problem, especially in the presence of strong hydrody-
namic forces, since binding forces are weak. Adsorption of enzymes may be stabilized by
cross-linking with glutaraldehyde. Glutaraldehyde treatment can denature some proteins.
Support materials used for enzyme adsorption can be inorganic materials, such as alumina,
silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials,
such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, and ion-exchange
resins, such as Amberlite, Sephadex, and Dowex. The surfaces of the support materials may
need to be pretreated (chemically or physically) for effective immobilization.
Covalent binding is the retention of enzymes on support surfaces by covalent bond
formation. Enzyme molecules bind to support material via certain functional groups, such
as amino, carboxyl, hydroxyl, and sulfhydryl groups. These functional groups must not be
in the active site. One common trick is to block the active site by flooding the enzyme
solution with a competitive inhibitor prior to covalent binding. Functional groups on sup-
port material are usually activated by using chemical reagents, such as cyanogen bromide,
carbodiimide, and glutaraldehyde. Support materials with various functional groups and
the chemical reagents used for the covalent binding of proteins are listed in Table 3.3.

80 Enzymes Chap. 3
 ch03

TABLE 3.3 Methods of Covalent Binding of Enzymes to Supports

10/11/01

Supports with —OH

(a) Using cyanogen bromide

11:08 PM
Page 81

(b) Using S-triazine derivatives

Supports with —NH2

(a) By diazotization

(b) Using glutaraldehyde

(continued )

81
 ch03
10/11/01

82
11:08 PM
Page 82

TABLE 3.3 Methods of Covalent Binding of Enzymes to Supports (Continued )

Supports with —COOH

(a) Via azide derivative

1)

2)

(b) Using a carbodiimide

Supports containing anhydrides

With permission, from D. I. C. Wang et al., Fermentation and Enzyme Technology, John Wiley & Sons, New York, 1979.
ch03 10/11/01 11:08 PM Page 83

Binding groups on the protein molecule are usually side groups (R) or the amino or car-
boxyl groups of the polypeptide chain.
The cross-linking of enzyme molecules with each other using agents such as glu-
taraldehyde, bis-diazobenzidine, and 2,2-disulfonic acid is another method of enzyme im-
mobilization. Cross-linking can be achieved in several different ways: enzymes can be
cross-linked with glutaraldehyde to form an insoluble aggregate, adsorbed enzymes may
be cross-linked, or cross-linking may take place following the impregnation of porous
support material with enzyme solution. Cross-linking may cause significant changes in
the active site of enzymes, and also severe diffusion limitations may result.
The most suitable support material and immobilization method vary depending on
the enzyme and particular application. Two major criteria used in the selection of support
material are (1) the binding capacity of the support material, which is a function of charge
density, functional groups, porosity, and hydrophobicity of the support surface, and
(2) stability and retention of enzymatic activity, which is a function of functional groups
on support material and microenvironmental conditions. If immobilization causes some
conformational changes on the enzyme, or if reactive groups on the active site of the en-
zyme are involved in binding, a loss in enzyme activity can take place upon immobiliza-
tion. Usually, immobilization results in a loss in enzyme activity and stability. However, in
some cases, immobilization may cause an increase in enzyme activity and stability due to
more favorable microenvironmental conditions. Because enzymes often have more than
one functional site that can bind the surface, an immobilized enzyme preparation may be
very heterogeneous. Even when binding does not alter enzyme structure, some enzyme
can be bound with the active site oriented away from the substrate solution and toward the
support surface, decreasing the access of the substrate to the enzyme. Retention of activity
varies with the method used. Table 3.4 summarizes the retention of activity of aminoacy-
lase immobilized by different methods.

TABLE 3.4 Effect of Immobilization Methods on the Retention of Enzymatic Activity

of Aminoacylase

Enzyme
Observed activity
activity immobilized
Support Method (units) (%)

Polyacrylamide Entrapment 526 52.6

Nylon Encapsulation 360 36.0
DEAE-cellulose Ionic binding 668 55.2
DEAE-Sephadex A-5O Ionic binding 680 56.2
CM-Sephadex C-5O Ionic binding 0 0
Iodoacetyl cellulose Covalent binding 472 39.0
CNBr-activated Sephadex Covalent binding 12 1.0
AE-cellulose Cross-linked with glutaraldehyde 8 0.6

With permission, from D. I. C. Wang et al., Fermentation and Enzyme Technology, John Wiley & Sons, New
York, 1979.

Sec. 3.4 Immobilized Enzyme Systems 83

ch03 10/11/01 11:08 PM Page 84

3.4.2. Diffusional Limitations in Immobilized

Enzyme Systems

Diffusional resistances may be observed at different levels in immobilized enzymes. These

resistances vary depending on the nature of the support material (porous, nonporous), hy-
drodynamical conditions surrounding the support material, and distribution of the enzyme
inside or on the surface of the support material. Whether diffusion resistance has a signifi-
cant effect on the rate of enzymatic reaction rate depends on the relative rate of the reaction
rate and diffusion rate, which is characterized by the Damköhler number (Da).
maximum rate of reaction Vm¢
Da = = (3.52)
maximum rate of diffusion k L [ Sb ]

where [Sb] is substrate concentration in bulk liquid (g/cm3) and kL is the mass-transfer co-
efficient (cm/s).
The rate of enzymatic conversion may be limited by diffusion of the substrate or re-
action, depending on the value of the Damköhler number. If Da >> 1, the diffusion rate is
limiting. For Da << 1, the reaction rate is limiting, and for Da ª 1, the diffusion and reac-
tion resistances are comparable. Diffusion and enzymatic reactions may be simultaneous,
with enzymes entrapped in a solid matrix, or may be two consecutive phenomena for ad-
sorbed enzymes.
3.4.2.1. Diffusion effects in surface-bound enzymes on nonporous sup-
port materials. Assume a situation where enzymes are bound and evenly distributed
on the surface of a nonporous support material, all enzyme molecules are equally active,
and substrate diffuses through a thin liquid film surrounding the support surface to reach
the reactive surfaces, as depicted in Fig. 3.17. Assume further that the process of immobi-
lization has not altered the protein structure, and the intrinsic kinetic parameters
(Vm, Km) are unaltered.

Figure 3.17. Substrate concentration pro-

file in a liquid film around adsorbed en-
zymes.

84 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 85

At steady state, the reaction rate is equal to the mass-transfer rate:

Vm¢ [Ss ]
( )
J s = k L [S b ] - [S s ] =
K m + [S s ]
(3.53)

where V¢m is the maximum reaction rate per unit of external surface area and kL is the liquid
mass-transfer coefficient. This equation is quadratic in [Ss], the substrate concentration at
the surface. It can be solved analytically, but the solution is cumbersome. Furthermore,
the value of [Ss] is not amenable to direct experimental observation.
Equation 3.53 can be solved graphically as depicted in Fig. 3.18. Such a plot also
makes it easy to visualize the effects of parameter changes such as stirring rate, changes in
bulk substrate concentration, or enzyme loading.

Figure 3.18. Graphical solution for amount of reaction per unit surface area for enzyme
immobilized on a nonporous catalyst. Curve A results from a knowledge of the intrinsic
solution-based kinetic parameters and the surface loading of enzyme (right side of
eq. 3.53). Line B is the mass transfer equation (left side of eq. 3.53). The intersection of
the two lines is the reaction rate, u, that can be sustained in the system. The responses for
two different bulk substrate concentrations are shown.

Sec. 3.4 Immobilized Enzyme Systems 85

ch03 10/11/01 11:08 PM Page 86

When the system is strongly mass-transfer limited, [Ss] ª 0, since the reaction is
rapid compared to mass transfer, and

v ª k L [Sb ], (for Da >> 1) (3.54)

and the system behaves as pseudo first order.
When the system is reaction limited (Da << 1), the reaction rate is often expressed as

V m¢ [Sb ]
v=
K m,app + [Sb ]
(3.55a)

where, with appropriate assumptions,

ÏÔ Vm¢ ¸Ô
K m,app = K m Ì1 + ˝ (3.55b)
(
ÓÔ k L [Sb ] + K m ) ˛Ô

Under these circumstances, the apparent Michaelis–Menten “constant” is a function of

stirring speed. Usually, Km,app is estimated experimentally as the value of [Sb], giving one-
half of the maximal reaction rate.

Example 3.4
Consider a system where a flat sheet of polymer coated with enzyme is placed in a stirred
beaker. The intrinsic maximum reaction rate (Vm) of the enzyme is 6 ¥ 10-6 mol/s-mg en-
zyme. The amount of enzyme bound to the surface has been determined to be 1 ¥ 10-4 mg
enzyme/cm2 of support. In solution, the value of Km has been determined to be 2 ¥ 10-3
mol/l. The mass-transfer coefficient can be estimated from standard correlations for stirred
vessels. We assume in this case a very poorly mixed system where kL = 4.3 ¥ 10-5 cm/s. What
is the reaction rate when (a) the bulk concentration of the substrate is 7 ¥ 10-3 mol/l? (b) Sb =
1 ¥ 10-2 mol/l?
Solution The solution is given in Fig. 3.18. The key is to note that the mass-transfer rate
equals the reaction rate at steady state, and as a consequence the right side of eq. 3.53 must
equal the left side. In case (a), this occurs at a substrate surface concentration of about 0.0015
mol/l with a reaction rate of 2.3 ¥ 10-10 mol/s-cm2. By increasing the bulk substrate concen-
tration to 0.01 mol/l, the value of [Ss] increases to 0.0024 mol/l with a reaction rate about
3.3 ¥ 10-10 mol/s-cm2.

3.4.2.2. Diffusion effects in enzymes immobilized in a porous matrix.

When enzymes are immobilized on internal pore surfaces of a porous matrix, substrate
diffuses through the tortuous pathway among pores and reacts with enzyme immobilized
on pore surfaces. Diffusion and reaction are simultaneous in this case, as depicted in
Fig. 3.19.
Assume that enzyme is uniformly distributed in a spherical support particle; the re-
action kinetics are expressed by Michaelis–Menten kinetics, and there is no partitioning
of the substrate between the exterior and interior of the support. Then we write the follow-
ing equation, stating that diffusion rate is equal to reaction rate at steady state:

86 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 87

Figure 3.19. Substrate concentration pro-

file in a porous support particle containing
immobilized enzymes. Here it is assumed
that no external substrate limitation exists so
that the bulk and surface concentrations are
the same.

Ê d 2 [S] 2 d[S] ˆ V m≤ [S]

De Á + = (3.56)
Ë dr
2 r dr ˜¯ K m + [S]

with boundary conditions [S] = [Ss] at r = R and d[S]/dr = 0 at r = 0, where Vm≤ is the max-
imum reaction rate per unit volume of support, and De is the effective diffusivity of sub-
strate within the porous matrix.
Equation 3.56 can be written in dimensionless form by defining the following di-
mensionless variables:

S=
[S] , r=
r
, b=
Km
[Ss ] R [S s ] (3.57a)
d 2 S 2 dS R 2 Vm≤ Ê S ˆ
+ = Á ˜
dr 2 r dr Ss De Ë S + b ¯

or
d 2 S 2 dS S
+ = 2 (3.57b)
dr 2 r dr 1+ S 

where

≤ Km
Vm
= R = Thiele modulus (3.57c)
De
_ _ _ _ _
With boundary conditions of S = 1 at r = 1 and dS/d r = 0 at r = 0, eq. 3.57 can be numer-
ically solved to determine the substrate profile inside the matrix. The rate of substrate
consumption is equal to the rate of substrate transfer through the external surface of the
support particle at steady state into the sphere.

d[S]
rs = Ns = 4 R 2 De (3.58)
dr r=R

Sec. 3.4 Immobilized Enzyme Systems 87

ch03 10/11/01 11:08 PM Page 88

Under diffusion limitations, the rate per unit volume is usually expressed in terms of
the effectiveness factor as follows:

≤ [S s ]
Vm (3.59)
rs = 
K m + [S s ]

The effectiveness factor is defined as the ratio of the reaction rate with diffusion
limitation (or diffusion rate) to the reaction rate with no diffusion limitation. The value of
the effectiveness factor is a measure of the extent of diffusion limitation. For h < 1, the
conversion is diffusion limited, whereas for h ª 1 values, conversion is limited by the re-
action rate and diffusion limitations are negligible. The factor is a function of f and b as
depicted in Figure 3.20.
For a zero-order reaction rate (b Æ 0), h ª 1 for a large range of Thiele modulus
values such as 1 < f < 100. For a first-order reaction rate (b Æ •), h = (f,b) and h is ap-
proximated to the following equation for high values of f.

3È 1 1˘
= Í - ˙ (3.60)
 Î tanh   ˚
When internal diffusion limits the enzymatic reaction rate, the rate-constant Vm,app
and Km,app values are not true intrinsic rate constants, but apparent values. To obtain true
intrinsic rate constants in immobilized enzymes, diffusion resistances should be elimi-
nated by using small particle sizes, a high degree of turbulence around the particles, and
high substrate concentrations.

Figure 3.20. Theoretical relationship between the effectiveness factor h and first-order
Thiele modulus, f, for a spherical porous immobilized particle for various values of b,
where b is the dimensionless Michaelis constant. (With permission, from D. I. C. Wang et
al., Fermentation and Enzyme Technology, John Wiley & Sons, Inc., New York, 1979,
p. 329.)

88 Enzymes Chap. 3
ch03 10/11/01 11:08 PM Page 89

Figure 3.21. The effectiveness factor decreases with increases in enzyme loading or
with increases in particle diameter. Point A represents the value of the effectiveness factor
for a particle radius of 10 mm with an enzyme loading of 100 mg/cm3, an enzyme activity
of 100 mmol/min per mg enzyme, a substrate diffusivity of 5 ¥ 10-6 cm2/s, and a bulk sub-
strate concentration tenfold higher than Km.

When designing immobilized enzyme systems using a particular support, the main
variables are Vm and R, since the substrate concentration, Km, and De are fixed. The
particle size (R) should be as small as possible within the constraints of particle integrity,
resistance to compression, and the nature of the particle recovery systems. The maximum
reaction rate is determined by enzyme activity and concentration in the support. High
enzyme content will result in high enzyme activity per unit of reaction volume but low
effectiveness factor. On the other hand, low enzyme content will result in lower en-
zyme activity per unit volume but a high effectiveness factor. For maximum conversion
rates, particle size should be small (Dp £ 10 mm) and enzyme loading should be
optimized. As depicted in the example in Fig. 3.21, Dp £ 10 mm and enzyme loadings
of less than 10 mg/cm3 are required for high values of the effectiveness factor
(h ≥ 0.8).

Example 3.5
D. Thornton and co-workers studied the hydrolysis of sucrose at pH = 4.5 and 25∞C using
crude invertase obtained from baker’s yeast in free and immobilized form. The following ini-
tial velocity data were obtained with 408 units of crude enzyme (1 unit = quantity of enzyme
hydrolyzing 1 mmol of sucrose/min when incubated with 0.29 M sucrose in a buffer at pH 4.5
and 25∞C).

Sec. 3.4 Immobilized Enzyme Systems 89

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V0 (mmol hydrolyzed/l-min)

Free enzyme Immobilized enzyme S0 (mol/l)

0.083 0.056 0.010

0.143 0.098 0.020
0.188 0.127 0.030
0.222 0.149 0.040
0.250 0.168 0.050
0.330 0.227 0.100
0.408 0.290 0.290

a. Determine the Km and Vm for this reaction using both free and immobilized enzyme.
b. Do the data indicate any diffusion limitations in the immobilized enzyme prepa-
ration?
Solution From a double-reciprocal plot of 1/v versus 1/S for free enzyme (Fig. 3.22),
-1/Km = -20 and Km = 0.05 M. 1/Vm = 2 and Vm = 0.5 mmol/l min. From a double-reciprocal
plot of 1/v versus 1/S for the immobilized enzyme, -1/Km = -20 and Km = 0.05 M. 1/Vm = 3
and Vm = 0.33 mmol/l-min. Since the Km values for free and immobilized enzymes are the
same, there is no diffusion limitation.

Figure 3.22. Double-reciprocal plots for free and immobilized enzymes (Example 3.4).

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3.4.3. Electrostatic and Steric Effects in Immobilized

Enzyme Systems

When enzymes are immobilized in a charged matrix as a result of a change in the mi-
croenvironment of the enzyme, the apparent bulk pH optimum of the immobilized en-
zyme will shift from that of soluble enzyme. The charged matrix will repel or attract
substrates, product, cofactors, and H+ depending on the type and quantity of surface
charge. For an enzyme immobilized onto a charged support, the shift in the pH-activity
profile is given by
zFy
DpH = pH i - pH e = 0.43 (3.61)
RT

where pHi and pHe are internal and external pH values, respectively; z is the charge
(valence) on the substrate; F is the Faraday constant (96,500 coulomb/eq. g); y is the
electrostatic potential; and R is the gas constant. Expressions similar to eq. 3.61 apply to
other nonreactive charged medium components. The intrinsic activity of the enzyme is al-
tered by the local changes in pH and ionic constituents. Further alterations in the apparent
kinetics are due to the repulsion or attraction of substrates or inhibitors.
The activity of an enzyme toward a high-molecular-weight substrate is usually re-
duced upon immobilization to a much greater extent than for a low-molecular-weight sub-
strate. This is mainly because of steric hindrance by the support. Certain substrates, such
as starch, have molecular weights comparable to those of enzymes and may therefore not
be able to penetrate to the active sites of immobilized enzymes.
Immobilization also affects the thermal stability of enzymes. Thermal stability often
increases upon immobilization due to the presence of thermal diffusion barriers and the
constraints on protein unfolding. However, decreases in thermal stability have been noted
in a few cases. The pH stability of enzymes usually increases upon immobilization, too.

3.5. LARGE-SCALE PRODUCTION OF ENZYMES

Among various enzymes produced at large scale are proteases (subtilisin, rennet), hydro-
lases (pectinase, lipase, lactase), isomerases (glucose isomerase), and oxidases (glucose
oxidase). These enzymes are produced using overproducing strains of certain organisms.
Separation and purification of an enzyme from an organism require disruption of cells, re-
moval of cell debris and nucleic acids, precipitation of proteins, ultrafiltration of the de-
sired enzyme, chromatographic separations (optional), crystallization, and drying. The
process scheme varies depending on whether the enzyme is intracellular or extracellular.
In some cases, it may be more advantageous to use inactive (dead or resting) cells with the
desired enzyme activity in immobilized form. This approach eliminates costly enzyme
separation and purification steps and is therefore economically more feasible. Details of
protein separations are covered in Chapter 11.
The first step in the large-scale production of enzymes is to cultivate the organisms pro-
ducing the desired enzyme. Enzyme production can be regulated and fermentation conditions
can be optimized for overproduction of the enzyme. Proteases are produced by using over-
producing strains of Bacillus, Aspergillus, Rhizopus, and Mucor; pectinases are produced by

Sec. 3.5 Large-Scale Production of Enzymes 91

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Figure 3.23. A flowsheet for the production of an extracellular enzyme.

Aspergillus niger; lactases are produced by yeast and Aspergillus; lipases are produced by
certain strains of yeasts and fungi; glucose isomerase is produced by Flavobacterium ar-
borescens or Bacillus coagulans. After the cultivation step, cells are separated from the media
usually by filtration or sometimes by centrifugation. Depending on the intracellular or extra-
cellular nature of the enzyme, either the cells or the fermentation broth is further processed to
separate and purify the enzyme. The recovery of intracellular enzymes is more complicated
and involves the disruption of cells and removal of cell debris and nucleic acids. Figure 3.23
depicts a schematic of an enzyme plant producing intracellular enzymes.
In some cases, enzyme may be both intracellular and extracellular, which requires
processing of both broth and cells. Intracellular enzymes may be released by increasing
the permeability of cell membrane. Certain salts such as CaCl2 and other chemicals such
as dimethylsulfoxide (DMSO) and pH shift may be used for this purpose. If enzyme re-
lease is not complete, then cell disruption may be essential.
The processes used to produce these industrial enzymes have much in common with
our later discussions on processes to make proteins from recombinant DNA.

3.6. MEDICAL AND INDUSTRIAL UTILIZATION OF ENZYMES

Enzymes have been significant industrial products for more than a hundred years. How-
ever, the range of potential application is increasing rapidly. With the advent of recombi-
nant DNA technology it has become possible to make formerly rare enzymes in large

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quantities and, hence, reduce cost. Also, in pharmaceutical manufacture the desire to
make chirally pure compounds is leading to new opportunities. Chirality is important in a
product; in a racemic mixture one enantiomer is often therapeutically useful while the
other may cause side effects and add no therapeutic value. The ability of enzymes to rec-
ognize chiral isomers and react with only one of them can be a key component in pharma-
ceutical synthesis. Processes that depend on a mixture of chemical and enzymatic
synthesis are being developed for a new generation of pharmaceuticals.
Technological advances have facilitated the use of enzymes over an increasingly
broad range of process conditions. Enzymes from organisms that grow in unusual environ-
ments (e.g., deep ocean, salt lakes, and hot springs) are increasingly available for study and
potential use. New enzymes and better control of reaction conditions allow the use of en-
zymes in the presence of high concentrations of organics, in high-salt aqueous environ-
ments, or at extreme temperatures, pH, or pressures. As we couple new insights into the
relationship of enzyme structure to biological function with recombinant DNA technology,
we are able to produce enzymes that are human designed or manipulated (see Section 14.9
on protein engineering). We no longer need to depend solely on natural sources for enzymes.
While there are many reasons to be optimistic about increasing use of enzymes, the
number of enzymes made at high volume for industrial purposes evolves more slowly. In
1996 the U.S. sales of industrial enzymes were $372 million, and sales are projected to
grow to $686 million by 2006. The products made in enzyme processes are worth billions
of dollars. Table 3.5 provides a breakdown of projected enzyme sales by industrial sector.
Table 3.6 lists some industrially important enzymes.
Proteases hydrolyze proteins into smaller peptide units and constitute a large and in-
dustrially important group of enzymes. Proteases constitute about 60% of the total
enzyme market. Industrial proteases are obtained from bacteria (Bacillus), molds (As-
pergillus, Rhizopus, and Mucor), animal pancreas, and plants. Most of the industrial pro-
teases are endoproteases. Proteases are used in food processing, such as cheese making
(rennet), baking, meat tenderization (papain, trypsin), and brewing (trypsin, pepsin); in
detergents for the hydrolysis of protein stains (subtilisin Carlsberg); and in tanning and
the medical treatment of wounds.

TABLE 3.5. Industrial Enzyme Market*

1996 Sales 2006 Projected Sales

Application (U.S. $ in millions) (U.S. $ in millions)

Food 170 214

Detergent 160 414
Textiles 27 32
Leather 11 13
Paper & Pulp 1 5
Other 3 8
TOTAL 372 686

*Data from C. Wrotnowski, Genetic & Engineering News, pp. 14 and

30, Feb. 1, 1997.

Sec. 3.6 Medical and Industrial Utilization of Enzymes 93