Mary K.

Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell

Chapter Six
The Behavior of Proteins: Enzymes

Paul D. Adams University of Arkansas

Enzyme Catalysis
Enzyme: a biological catalyst
with the exception of some RNAs that
catalyze their own splicing (Section 10.4), all
enzymes are proteins
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, G
an enzyme provides an alternative pathway
with a lower activation energy

Enzyme Catalysis (Contd)

For a reaction taking place at constant temperature
and pressure, e.g., in the body
A

the change in free energy is

G = H - TS

Difference in energies between initial state and

final state
G = RT ln K eq

The change in free energy is related to the

equilibrium constant, Keq, for the reaction by

Enzyme Catalysis (Contd)

Consider the reaction
H2 O2

H2 O +

O2

Temperature dependence of catalysis

Temperature can also
catalyze reaction (increase
rate)
This is dangerous, why?
Increasing temperature will
eventually lead to protein
denaturation

Enzyme Kinetics
For the reaction

A + B

The rate of reaction is given by rate equation

[A]
[B]
_
_
Rate =
=
t
t

[P]
=
t

Rate = k[A] f[B]g

Where k is a proportionality constant called the

specific rate constant
Order of reaction: the sum of the exponents in the
rate equation

Enzyme Kinetics (Contd)

Consider the reaction

A + B

C + D

Whose rate equation is given by the expression

Rate = k[A] 1[B]1

Determined experimentally, not always from balanced

equations

The reaction is said to be first order in A, first order in

B, and second order overall
Consider this reaction of glycogen with phosphate
Glycogen n + HPO 42-

Glucose-1-phosphate + Glycogen

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

24 ]

n-1

How Enzymes bind to Substrate

In an enzyme-catalyzed reaction
Substrate, S: a reactant
Active site: the small portion of the enzyme surface
where the substrate(s) becomes bound by noncovalent
forces, e.g., hydrogen bonding, electrostatic
attractions, van der Waals attractions
E + S

ES
enzyme-substrate
complex

Binding Models
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

2 Modes of E-S Complex Formation

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

An Example of Enzyme Catalysis (Contd)

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

Sigmoidal shape- characteristic of allosterism
Again Max. velocity reached, but different mechanism

Michaelis-Menten Kinetics
Initial rate of an enzyme-catalyzed reaction versus
substrate concentration

Michaelis-Menten Model
For an enzyme-catalyzed reaction
E + S

k1
k-1

ES

k2

The rates of formation and breakdown of ES are

given by these equations
rate of formation of ES = k1 [E][S]
rate of breakdown of ES = k -1 [ES] + k 2[ES]

At the steady state

k1 [E][S] = k -1[ES] + k 2[ES]

Michaelis-Menten Model (Contd)

When the steady state is reached, the concentration
of free enzyme is the total less that bound in ES
[E] = [E] T - [ES]

Substituting for the concentration of free enzyme and

collecting all rate constants in one term gives
([E]T - [ES]) [S]
[ES]

k-1 + k2
k1

= KM

Where KM is called the Michaelis constant

Michaelis-Menten Model (Contd)

It is now possible to solve for the concentration of the
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
[ES]

= KM

[E]T [S] - [ES][S] = KM[ES]

[E]T [S] = [ES](K M + [S])

Or alternatively

[ES]

[E] T [S]
=
KM + [S]

Michaelis-Menten Model (Contd)

In the initial stages, formation of product depends only on the
rate of breakdown of ES

Vinit = k2 [ES]

k 2[E]T [S]
=
KM + [S]

If substrate concentration is so large that the enzyme is

saturated with substrate [ES] = [E]T

Vinit = Vmax = k2 [E]T

Substituting k2[E]T = Vmax into the top equation gives

Vinit =

Vmax [S]
KM + [S]

Michaelis-Menten
equation

Michaelis-Menten Model (Contd)

When [S]= KM, the equation reduces to

V=

Vmax [S]
KM + [S]

Vmax [S]
[S] + [S]

Vmax
2

Linearizing The Michaelis-Menten Equation

It is difficult to determine Vmax experimentally
The equation for a hyperbola

Vmax [S]
V=
KM + [S]

(an equation for a hyperbola)

Can be transformed into the equation for a straight line by taking

the reciprocal of each side

1 =
V

KM + [S]
Vmax [S]

KM

Vmax [S]

KM
1 =
+ 1
V
Vmax [S]
Vmax

[S]
Vmax [S]

Lineweaver-Burk Plot
The Lineweaver-Burke plot has the form y = mx + b, and is the
formula for a straight line

1
V

KM
=
Vmax

[S]

1
Vmax

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

Lineweaver-Burk Plot (Contd)

KM is the dissociation constant for ES; the greater the value of
KM, the less tightly S is bound to E
Vmax is the turnover number

Turnover Numbers
Vmax is related to the turnover number of enzyme:also
called kcat

V max

turnover _ number kcat

[ET ]
Number of moles of substrate that react to form product
per mole of enzyme per unit of time

Enzyme Inhibition

Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but

can be released

competitive inhibitor: binds to the active (catalytic) site and

blocks access to it by substrate
noncompetitive inhibitor: binds to a site other than the active site;
inhibits the enzyme by changing its conformation

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
Substrate must compete with inhibitor for the active
site; more substrate is required to reach a given
reaction velocity

EI

+ E + S

ES

We can write a dissociation constant, KI for EI

EI

+ E

KI =

[E][I]
[EI]

Competitive Inhibition

Competitive Inhibition
No inhibition
1 = KM
V
Vmax
y =

1
S

Vmax
b

In the presence of a competitive inhibitor

1 = KM 1 + [I]
V
Vmax
KI

1
S

Vmax
b

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

versus 1/[S], the slope (and the x intercept) changes
but the y intercept does not change

A Lineweaver-Burke Plot for Competitive

Inhibition

Noncompetitive Inhibition (Contd)

Several equilibria are involved
+S

E
-I

ES

-S
+I

-I
+S

EI

-S

E + P

+I

ESI

The maximum velocity Vmax has the form

I

max =

Vmax
1 + [I]/KI

Noncompetitive Inhibition (Contd)

A Lineweaver-Burke Plot for

Noncompetitive Inhibition

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
V
S
Vmax

Vmax

y =

m x + b
In the presence of a noncompetitive inhibitor

1 = KM 1 + [I]
V
Vmax
KI

1
S

Vmax

[I]
1 +
KI
b

A Lineweaver-Burke Plot for

Noncompetitive Inhibition (Contd)

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.