Enzyme Catalysis

Mary K. Campbell • Enzyme: a biological catalyst

Shawn O. Farrell • with the exception of some RNAs
that catalyze their own splicing
international.cengage.com/
(Section 10.4), all enzymes are
proteins
The Behavior of Proteins: Enzymes • enzymes can increase the rate of
a reaction by a factor of up to
1020 over an uncatalyzed reaction
• some enzymes are so specific
that they catalyze the reaction of
only one stereoisomer; others
catalyze a family of similar
reactions
• The rate of a reaction depends on its
activation energy, DG°‡
• an enzyme provides an
alternative pathway with a lower
Paul D. Adams • University of Arkansas activation energy

Enzyme Catalysis (Cont’d) Enzyme Catalysis (Cont’d)

• For a reaction taking place at constant temperature • Consider the reaction
and pressure, e.g., in the body
A B
H2 O 2 H2 O + O2
• the change in free energy is
DG° = DH° - TDS°
• Difference in energies between initial state and
final state
DG° = -RT ln Keq

• The change in free energy is related to the

equilibrium constant, Keq, for the reaction by

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Temperature dependence of catalysis Enzyme Kinetics
• Temperature can also • For the reaction
A + B P
catalyze reaction (increase
rate) • The rate of reaction is given by rate equation

• This is dangerous, why? Rate = _ D[A] = _ D[B] = D[P]

Dt Dt Dt
Rate = k[A]f[B]g
• Increasing temperature will
eventually lead to protein • Where k is a proportionality constant called the
denaturation specific rate constant
• Order of reaction: the sum of the exponents in the
rate equation

Enzyme Kinetics (Cont’d) How Enzymes bind to Substrate

• Consider the reaction • In an enzyme-catalyzed reaction
A+ B C + D • Substrate, S: a reactant
Whose rate equation is given by the expression • Active site: the small portion of the enzyme surface
Rate = k[A]1[B]1 where the substrate(s) becomes bound by noncovalent
forces, e.g., hydrogen bonding, electrostatic
• Determined experimentally, not always from balanced attractions, van der Waals attractions
equations
• The reaction is said to be first order in A, first order in
B, and second order overall E + S ES
enzyme-subs trate
• Consider this reaction of glycogen with phosphate complex
Glycogenn + HPO42- Glucose-1-phosphate + Glycogenn-1

Rate = k[Glycogen]1[HPO 42-]1 = k[Glycogen][HPO42-]

2
Binding Models 2 Modes of E-S Complex Formation
• Two models have been developed to describe
formation of the enzyme-substrate complex

• Lock-and-key model: substrate binds to that portion

of the enzyme with a complementary shape

• Induced fit model: binding of the substrate induces a

change in the conformation of the enzyme that results
in a complementary fit

Formation of Product An Example of Enzyme Catalysis

• Chymotrypsin catalyzes
• The selective hydrolysis of peptide bonds where the
carboxyl is contributed by Phe and Tyr

• It also catalyzes hydrolysis of the ester bonds

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An Example of Enzyme Catalysis (Cont’d) Non-Allosteric Enzyme Behavior
• Point at which the rate of
reaction does not change,
enzyme is saturated,
maximum rate of reaction
is reached

ATCase: An Example of Allosteric Behavior Michaelis-Menten Kinetics

• Sigmoidal shape- characteristic of allosterism • Initial rate of an enzyme-catalyzed reaction versus
substrate concentration
• Again Max. velocity reached, but different mechanism

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Michaelis-Menten Model Michaelis-Menten Model (Cont’d)
• For an enzyme-catalyzed reaction • When the steady state is reached, the concentration
k1
of free enzyme is the total less that bound in ES
k2
E + S ES P
k-1
[E] = [E]T - [ES]
• The rates of formation and breakdown of ES are
given by these equations • Substituting for the concentration of free enzyme and
collecting all rate constants in one term gives
rate of f ormation of ES = k1[E][S] ([E]T - [ES]) [S ] k-1 + k2
= = KM
rate of breakdown of ES = k-1 [ES] + k2[ES] [ES] k1

• At the steady state • Where KM is called the Michaelis constant

k1[E][S] = k-1[ES] + k2[ES]

Michaelis-Menten Model (Cont’d) Michaelis-Menten Model (Cont’d)

• It is now possible to solve for the concentration of the • When [S]= KM, the equation reduces to
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
= KM
[ES]
Vmax [S] Vmax [S] Vmax
[E]T [S] - [ES][S] = KM[ES] V= = =
KM + [S] [S] + [S] 2
[E]T [S] = [ES](K M + [S])

• Or alternatively [E] T [S]

[ES] =
KM + [S]

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Michaelis-Menten Model (Cont’d) Linearizing The Michaelis-Menten Equation
• In the initial stages, formation of product depends only on the • It is difficult to determine Vmax experimentally
rate of breakdown of ES • The equation for a hyperbola

k 2[E]T [S] Vmax [S]

Vinit = k2[ES] = V= (an equation for a hyperbola)
KM + [S] KM + [S]
• If substrate concentration is so large that the enzyme is • Can be transformed into the equation for a straight line by taking
saturated with substrate [ES] = [E]T the reciprocal of each side

Vinit = Vmax = k2[E]T

1 = KM + [S] KM [S]
• Substituting k2[E]T = Vmax into the top equation gives = +
V Vmax [S] Vmax [S] Vmax [S]

Vmax [S] Michaelis-Menten KM

Vinit =
equation
1 = + 1
KM + [S] V Vmax [S] Vmax

Lineweaver-Burk Plot Lineweaver-Burk Plot (Cont’d)

• The Lineweaver-Burke plot has the form y = mx + b, and is the • KM is the dissociation constant for ES; the greater the value of
formula for a straight line KM, the less tightly S is bound to E
1 = KM 1 + 1
•
V Vmax [S ] Vmax • Vmax is the turnover number
y = m • x + b
• a plot of 1/V versus 1/[S] will give a straight line with slope of
KM/Vmax and y intercept of 1/Vmax
• such a plot is known as a Lineweaver-Burk double reciprocal
plot

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Turnover Numbers Enzyme Inhibition
• Reversible inhibitor: a substance
• Vmax is related to the turnover number of enzyme:also that binds to an enzyme to inhibit it,
called kcat but can be released
• competitive inhibitor: binds to
æ V max ö the active (catalytic) site and
ç ÷ = turnover _ number = kcat blocks access to it by
è [ET ] ø substrate
• noncompetitive inhibitor: binds
• Number of moles of substrate that react to form product to a site other than the active
site; inhibits the enzyme by
per mole of enzyme per unit of time changing its conformation
• Uncompetitive inhibitor: Binds
to the complex
• Irreversible inhibitor: a substance
that causes inhibition that cannot be
reversed
• usually involves formation or
breaking of covalent bonds to
or on the enzyme

Competitive Inhibition Competitive Inhibition

• Substrate must compete with inhibitor for the active No inhibition
site; more substrate is required to reach a given 1 = KM • 1 + 1
reaction velocity V Vmax S Vmax
y = m • x + b
In the pres ence of a com petitive inhibitor
EI I + E + S ES P
1 = KM 1 + [I] 1 + 1
• We can write a dissociation constant, KI for EI V Vmax KI S Vmax

y = m • x + b

• In a Lineweaver-Burk double reciprocal plot of 1/V

EI I + E
[E][I] versus 1/[S], the slope (and the x intercept) changes
KI =
[EI] but the y intercept does not change

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A Lineweaver-Burke Plot for Competitive
Noncompetitive Inhibition (Cont’d)
Inhibition
• Several equilibria are involved

+S
E ES E + P
-S
-I +I -I +I

+S
EI ESI
-S

• The maximum velocity Vmax has the form

Vmax
VImax =
1 + [I]/KI

A Lineweaver-Burke Plot for A Lineweaver-Burke Plot for

Noncompetitive Inhibition Noncompetitive Inhibition (Cont’d)
• Because the inhibitor does not interfere with binding of substrate
to the active site, KM is unchanged
• Increasing substrate concentration cannot overcome
noncompetitive inhibition

No inhibition
1 = KM • 1 + 1
V Vmax S Vmax
y = m • x + b
In t he presence of a noncompetitive i nhibitor
1 = KM 1 + [I] 1 + 1
1 +
[I]
V Vma x KI S Vma x KI

y = m • x + b

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Other Types of Inhibition
• Uncompetitive- inhibitor can bind to the ES complex
but not to free E. Vmax decreases and KM decreases.

• Mixed- Similar to noncompetitively, but binding of I

affects binding of S and vice versa.