Properties of Enzymes

• Catalyst - speeds up attainment of reaction

equilibrium
• Enzymatic reactions - 103 to 1017 faster than
the corresponding uncatalyzed reactions
• Substrates - highly specific reactants for
enzymes

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 Properties of enzymes (continued)

• Stereospecificity - many enzymes act upon

only one stereoisomer of a substrate
• Reaction specificity - enzyme product yields
are essentially 100% (there is no formation of
wasteful byproducts)
• Active site - where enzyme reactions take
place
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 The Six Classes of Enzymes

1. Oxidoreductases (dehydrogenases)
• Catalyze oxidation-reduction reactions

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 2. Transferases

• Catalyze group transfer reactions

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 3. Hydrolases

• Catalyze hydrolysis reactions where water is

the acceptor of the transferred group

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 4. Lyases

• Catalyze lysis of a substrate, generating a

double bond in a nonhydrolytic, nonoxidative
elimination (Synthases catalyze the addition to
a double bond, the reverse reaction of a lyase)

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 5. Isomerases

• Catalyze isomerization reactions

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 6. Ligases (synthetases)

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)

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 Kinetic Experiments Reveal
Enzyme Properties

A. Chemical Kinetics
• Experiments examine the amount of product
(P) formed per unit of time (∆ [P] / ∆ t)
• Velocity (v) - the rate of a reaction
(varies with reactant concentration)
• Rate constant (k) - indicates the speed or
efficiency of a reaction

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 First order rate equation

• Rate for nonenzymatic conversion of substrate

(S) to product (P) in a first order reaction:
(k is expressed in reciprocal time units (s-1 ))

∆ [P] / ∆ t = v = k[S]

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 Second order reaction

• For reactions: S1 + S2 P1 + P2
• Rate is determined by the concentration
of both substrates
• Rate equation: v = k[S1]1[S2]1

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 Pseudo first order reaction

• If the concentration of one reactant is so high

that it remains essentially constant, reaction
becomes zero order with respect to that reactant
• Overall reaction is then pseudo first-order

v = k[S1]1[S2]0 = k’[S1]1

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 B. Enzyme Kinetics

• Enzyme-substrate complex (ES) - complex

formed when specific substrates fit into the enzyme
active site
E + S ES E+P

• When [S] >> [E], every enzyme binds a molecule

of substrate (enzyme is saturated with substrate)
• Under these conditions the rate depends only
upon [E], and the reaction is pseudo-first order

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 Effect of enzyme concentration [E]
on velocity (v)

• Fixed, saturating [S]

• Pseudo-first order
enzyme-catalyzed
reaction

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 Initial velocity (vo)

• Velocity at the beginning of an enzyme-catalyzed

reaction is vo (initial velocity)

• k1 and k-1 represent rapid noncovalent

association /dissociation of substrate from enzyme
active site
• k2 = rate constant
k for formation of product from ES
1 k2
E+S ES E+P
k-1

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 Progress curve for an
enzyme-catalyzed reaction

• The initial velocity (vo)

is the slope of the initial
linear portion of the
curve
• Rate of the reaction
doubles when twice as
much enzyme is used

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 The Michaelis-Menten Equation

• Maximum velocity (Vmax ) is reached when an

enzyme is saturated with substrate (high [S])
• At high [S] the reaction rate is independent of
[S] (zero order with respect to S)
• At low [S] reaction is first order with respect to S
• The shape of a vo versus [S] curve is a
rectangular hyperbola, indicating saturation of
the enzyme active site as [S] increases
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 Plots of initial velocity
(vo) versus [S]

(a) Each vo vs [S] point is

from one kinetic run
(b) Michaelis constant (Km)
equals the concentration
of substrate needed for
1/2 maximum velocity

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 The Michaelis-Menten equation

• Equation describes vo versus [S] plots

• Km is the Michaelis constant

Vmax [S]
vo =
Km + [S]

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 A. Derivation of the
Michaelis-Menten Equation

• Derived from
(1) Steady-state conditions:
Rate of ES formation = Rate of ES decomposition
(2) Michaelis constant: Km = (k-1 + k2) / k1
(3) Velocity of an enzyme-catalyzed reaction
(depends upon rate of conversion of ES to E + P)
vo = k2[ES]
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 B. The Meanings of Km

• Km = [S] when vo = 1/2 Vmax

• Km ≅ k-1 / k1 = Ks (the enzyme-substrate
dissociation constant) when kcat << either k1 or k-1

• The lower the value of Km, the tighter the

substrate binding
• Km can be a measure of the affinity of E for S
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 Kinetic Constants Indicate Enzyme
Activity and Specificity

• Catalytic constant (kcat ) - first order rate

constant for conversion of ES complex to E + P
• kcat most easily measured when the enzyme is
saturated with S (region A on Figure 5)
• Ratio kcat /Km is a second order rate constant for
E+S E + P at low [S] concentrations
(region B on Figure 5)
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 Meanings of kcat and kcat /Km

Rate of Conversion

Catalytic Efficiency

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 Values of kcat /Km

• kcat /Km can approach rate of encounter of two

uncharged molecules in solution (108 to 109M-1 s-1 )
• kcat /Km is also a measure of enzyme specificity for
different substrates (specificity constant)
• rate acceleration = kcat /kn
(kn = rate constant in the absence of enzyme)

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 Measurement of Km and Vmax

Fig 5.6 The double-

reciprocal Lineweaver-
Burk plot is a linear
transformation of the
Michaelis-Menten plot
(1/vo versus 1/[S])

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 Reversible Enzyme Inhibition

• Inhibitor (I) binds to an enzyme and prevents

formation of ES complex or breakdown to E + P
• Inhibition constant (Ki) is a dissociation constant
EI E+I
• There are three basic types of inhibition:
Competitive, Uncompetitive and Noncompetitive
• These can be distinguished experimentally by their
effects on the enzyme kinetic patterns
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 Reversible
enzyme inhibitors

(a) Competitive. S and I

bind to same site on E
(b) Nonclassical
competitive. Binding of
S at active site prevents
binding of I at separate
site. Binding of I at
separate site prevents S
binding at active site.
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 Reversible
enzyme
inhibitors(cont)
(c) Uncompetitive.
I binds only to ES
(inactivates E)
(d) Noncompetitive.
I binds to either E or
ES to inactivate the
enzyme

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 A. Competitive Inhibition

• Inhibitor binds only to free enzyme (E) not (ES)

• Substrate cannot bind when I is bound at active site
(S and I “compete” for the enzyme active site)
• Vmax is the same with or without I (high S can still
saturate the enzyme even in the presence of I)
• Apparent Km (Kmapp ) measured in the presence of I is
larger than KM (measured in absence of I)
• Competitive inhibitors usually resemble the substrate
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 Competitive inhibition.
(a) Kinetic scheme. (b) Lineweaver-Burk
plot

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 B. Uncompetitive Inhibition

• Uncompetitive inhibitors bind to ES not to free E

• Vmax decreased by conversion of some E to ESI
• Km (Kmapp ) is also decreased
• Lines on double-reciprocal plots are parallel
• This type of inhibition usually only occurs in
multisubstrate reactions
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 Uncompetitive inhibition

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 C. Noncompetitive Inhibition

• Noncompetitive inhibitors bind to both E and ES

• Inhibitors do not bind at the same site as S
• Vmax decreases
• Km does not change
• Inhibition cannot be overcome by addition of S
• Lines on double-reciprocal plot intersect on x axis
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 Noncompetitive inhibition

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 Irreversible Enzyme Inhibition

• Irreversible inhibitors form stable covalent bonds

with the enzyme (e.g. alkylation or acylation of an
active site side chain)
• There are many naturally-occurring and synthetic
irreversible inhibitors
• These inhibitors can be used to identify the amino
acid residues at enzyme active sites
• Incubation of I with enzyme results in loss of activity
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 Inhibition of serine protease with DFP

• Diisopropyl fluorophosphate (DFP) is an organic

phosphate that inactivates serine proteases
• DFP reacts with the active site serine (Ser-195) of
chymotrypsin to form DFP-chymotrypsin
• Such organophosphorous inhibitors are used as
insecticides or for enzyme research
• These inhibitors are toxic because they inhibit
acetylcholinesterase (a serine protease that
hydrolyzes the neurotransmitter acetylcholine)
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• Reaction of DFP with
Ser-195 of chymotrypsin

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 Coenzymes and Vitamins

• Some enzymes require cofactors for activity

(1) Essential ions (mostly metal ions)
(2) Coenzymes (organic compounds)

Apoenzyme + Cofactor Holoenzyme

(protein only) (active)
(inactive)
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 Coenzymes

• Coenzymes act as group-transfer reagents

• Hydrogen, electrons, or other groups can be
transferred
• Larger mobile metabolic groups can be attached
at the reactive center of the coenzyme
• Coenzyme reactions can be organized by their
types of substrates and mechanisms
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 Types of cofactors

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 Many Enzymes Require Inorganic Cations

• Enzymes requiring metal ions for full activity:

(1) Metal-activated enzymes have an absolute
requirement or are stimulated by metal ions
(examples: K+, Ca2+ , Mg2+ )
(2) Metalloenzymes contain firmly bound metal
ions at the enzyme active sites (examples:
iron, zinc, copper, cobalt )

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 Protein Coenzymes

• Protein coenzymes (group-transfer proteins) contain

a functional group as part of a protein or as a
prosthetic group
• Participate in:
(1) Group-transfer reactions
(2) Oxidation-reduction reactions where transferred
group is a hydrogen or an electron
• Metal ions, iron-sulfur clusters and heme groups are
commonly found in these proteins
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 Coenzyme Classification

• There are two classes of coenzymes

(1) Cosubstrates are altered during the reaction
and regenerated by another enzyme
(2) Prosthetic groups remain bound to the
enzyme during the reaction, and may be
covalently or tightly bound to enzyme

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 Classification of coenzymes in mammals

(1) Metabolite coenzymes - synthesized from

common metabolites
(2) Vitamin-derived coenzymes - derivatives of
vitamins (vitamins cannot be synthesized by
mammals, but must be obtained as
nutrients)

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 A. Metabolite Coenzymes

• Nucleoside triphosphates are examples

ATP

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 Reactions of ATP

• ATP is a versatile reactant that can donate its:

(1) Phosphoryl group (γ -phosphate)
(2) Pyrophosphoryl group (γ ,β phosphates)
(3) Adenylyl group (AMP)
(4) Adenosyl group

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B. Vitamin-Derived Coenzymes and Nutrition

• Vitamins are required for coenzyme synthesis

and must be obtained from nutrients
• Animals rely on plants and microorganisms for
vitamin sources (meat supplies vitamins also)
• Most vitamins must be enzymatically
transformed to the coenzyme

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Oxidized, reduced forms of NAD (NADP)

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 NAD and NADP are cosubstrates
for dehydrogenases

• Oxidation by pyridine nucleotides always occurs

two electrons at a time
• Dehydrogenases transfer a hydride ion (H:-) from a
substrate to pyridine ring C-4 of NAD+ or NADP+
• The net reaction is:
NAD(P)+ + 2e- + 2H+ NAD(P)H + H+

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 FAD and FMN

• Flavin adenine dinucleotide (FAD) and Flavin

mono-nucleotide (FMN) are derived from
riboflavin (Vit B2)
• Flavin coenzymes are involved in oxidation-
reduction reactions for many enzymes
(flavoenzymes or flavoproteins)
• FAD and FMN catalyze one or two electron
transfers
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Fig 7.12 Reduction, reoxidation of FMN or FAD

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 Coenzyme A (CoA or HS-CoA)

• Derived from the vitamin pantothenate (Vit B3)

• Participates in acyl-group transfer reactions with
carboxylic acids and fatty acids
• CoA-dependent reactions include oxidation of
fuel molecules and biosynthesis of carboxylic
acids and fatty acids
• Acyl groups are covalently attached to the -SH
of CoA to form thioesters
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 Coenzyme A

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 Ubiquinone (Coenzyme Q)

• Found in respiring organisms and

photosynthetic bacteria
• Transports electrons between membrane-
embedded complexes
• Plastoquinone (ubiquinone analog) functions in
photosynthetic electron transport

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 (a) Ubiquinone,
(b) Plastoquinone

• Hydrophobic tail of each is composed of 6 to 10

five-carbon isoprenoid units
• The isoprenoid chain allows these quinones to
dissolve in lipid membranes

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 Cytochromes

• Heme-containing coenzymes whose Fe(III)

undergoes reversible one-electron reduction
• Cytochromes a,b and c have different visible
absorption spectra and heme prosthetic groups
• Electron transfer potential varies among
different cytochromes due to the different
protein environment of each prosthetic group

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 (a) Heme group of cyt a

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 (b) Heme group of cyt b

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 (c) Heme group of cyt c

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 Regulation
Temperature vs. Enzyme Activity

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 pH vs. Enzyme Activity

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 Regulation of Enzyme Activity

• Regulatory enzymes - activity can be reversibly

modulated by effectors
• Such enzymes are usually found at the first unique
step in a metabolic pathway (the first “committed”
step)
• Regulation at this step conserves material and
energy and prevents accumulation of intermediates

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 Two Methods of regulation

(1) Noncovalent allosteric regulation

(2) Covalent modification
• Allosteric enzymes have a second
regulatory site (allosteric site) distinct from
the active site
• Allosteric inhibitors or activators bind to this
site and regulate enzyme activity via
conformational changes
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 A. Regulation by Covalent Modification

• Interconvertible enzymes are controlled by

covalent modification
• Converter enzymes catalyze covalent
modification
• Converter enzymes are usually controlled
themselves by allosteric modulators

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 General scheme for covalent modification
of interconvertible enzymes

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 Pyruvate dehydrogenase regulation

• Phosphorylation
stabilizes the inactive
state (red)
• Dephosphorlyation
stabilizes the active
state (green)

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 B. Phosphofructokinase-1 (PFK-1)
Is an Allosteric Enzyme

• PFK-1catalyzes an early step in glycolysis

(an ATP-generating pathway for glucose
degradation)
• Phosphoenol pyruvate (PEP), an intermediate
near the end of the pathway is an allosteric
inhibitor of PFK-1
• ADP is an allosteric activator of PFK-1

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 Regulation of glycolysis by PFK-1

• When the ratio [PEP]/[ADP] is high, flux through

glycolysis decreases at the PFK-1 step
• When the ratio of [PEP]/[ADP] is low, PFK-1 is
activated and glycolysis produces more ATP
from ADP
• Thus the concentrations of PEP and ADP act
allosterically through PFK-1 to regulate the
activity of the entire pathway
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 Reaction catalyzed by PFK-1

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 Phosphoenolpyruvate (PEP)

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 Effect of PEP and ADP on PFK-1

• Both PEP and ADP affect the binding of the

substrate fructose 6-phosphate (F6P) to PFK-1
• PFK-1 exhibits a sigmoidal (S-shaped) vo versus [S]
curve. This is due to the cooperativity of S binding.
• Increasing ADP concentrations lowers the apparent
Km for F6P without affecting Vmax (activates PFK)

• The allosteric inhibitor PEP raises the apparent Km

without changing Vmax
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 Plots of initial velocity versus
F6P for PFK-1

• ADP is an allosteric
activator of PFK-1
and lowers the
apparent Km without
affecting Vmax
• For a given F6P
concentration the vo
is larger in the
presence of ADP
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C. General Properties of Allosteric Enzymes

1. Activities of allosteric regulator enzymes are

changed by inhibitors and activators (modulators)
2. Allosteric modulators bind noncovalently to the
enzymes that they regulate
3. Regulatory enzymes possess quaternary
structure
(continued next slide)

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General properties of regulatory enzymes (cont)

4. Regulatory enzymes have at least one substrate

which has a sigmoidal vo versus [S] curve (positive
cooperativity of multiple substrate binding sites)
5. There is a rapid transition between the active (R)
and inactive (T) conformations
6. Substrates and activators may bind only to the R
state while inhibitors may bind only to the T state

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 Rapid transition exists between R and T states

• Addition of S increases concentration of the R state

• Addition of I increases concentration of the T state
• Activator molecules bind preferentially to R, leading
to an increase in the R/T ratio

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 Role of cooperativity of
binding in regulation

• Addition of modulators alters enzyme activity

• Activators can lower Km, inhibitors can raise Km

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 D. Two Theories of Allosteric Regulation

Concerted theory - (symmetry-driven theory).

• Only 2 conformations exist: R and T ( symmetry is
retained in the shift between R and T states)
• Subunits are either all R or all T
• R has high affinity for S, T has a low affinity for S
• Binding of S shifts the equilibrium toward all R state
• Binding of I shifts the equilibrium toward all T state
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 Sequential Theory (ligand-induced theory)

• A ligand may induce a change in the structure

of the subunit to which it binds
• Conformational change of one subunit may
affect the conformation of neighboring subunits
• A mixture of both R (high S affinity) and T (low
S affinity) subunits may exist (symmetry does
not have to be conserved)

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 Two models

(a) Concerted model:

subunits either all T state
or all R state
(b) Sequential model:
Mixture of T subunits and
R subunits is possible.
Binding of S converts
only that subunit from T
to R
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