6 | Enzymes

2009 W. H. Freeman and Company

CHAPTER 6
Enzymes
Key topics :
Physiological significance of enzymes
Origin of catalytic power of enzymes
Description of enzyme kinetics and inhibition
Chemical mechanisms of catalysis
Mechanisms of chymotrypsin, enolase, and
lysozyme

Why Use Proteins as

Biocatalysts?

Higher reaction rates

Greater reaction specificity
Milder reaction conditions
Capacity for regulation

COO

COO

NH2

O
OH

COO

O
COO

OH

COO

COO

Chorismate
mutase

COO

OOC

NH2

OH

Metabolites have
many potential
pathways of
alteration
Enzymes make the
desired one most
favorable

What are Enzymes?

Enzymes are catalytically active biological
macromolecules
Most enzymes are globular proteins, however
some RNA also catalyze reactions (ribozymes)
Study of enzymatic processes is the oldest field of
biochemistry, dating back to late 1700s
Study of enzymes has dominated biochemistry in
the past and continues to do so

What Do Enzymes Do?

Under biologically relevant conditions, uncatalyzed
reactions tend to be slow
An enzyme catalyzes reactions by providing a
specific environment (active site) within which a
given reaction can proceed more rapidly
The molecule that is catalyzed is referred to as the
substrate
The active site is lined with residues that bind the
substrate and catalyze its chemical transformation
Active site residues also contribute to substrate
specificity

Enzymatic Substrate Specificity

OH

H
-

H
+
NH3

OOC

OOC

H
-

No binding

+
NH3

OOC

+
NH3

OH
HO

OH

H
H

H
NH

Binding but no reaction

CH3

Example: Phenylalanine hydroxylase

Enzyme-Substrate Complex
ES complex is central to enzyme action (and
mathematical treatments of enzyme catalyzed
reactions)
Enzymes act by binding substrates
the enzyme substrate complex is known as the
Michaelis complex
allows thinking in terms of chemical
interactions
allows development of kinetic equations

vmax [ S ]
Km [S ]

Transition State Theory

Slow reactions face significant activation
barriers that must be surmounted during the
reaction (an energy hill)
The top of the energy hill is the transition
state
The transition state is a fleeting molecular
moment in which events such as bond
breakage/formation or charge formation have
proceeded to the point where decay to either
substrate or product is equally likely.

Transition State Theory

The difference between the energy levels of the
ground state (S or P) and transition state is the
activation energy (G)
The rate of the reaction reflects the G, the
higher the G the slower the reaction
The reaction equilibrium is related to the free
energy change for the reaction (Go); a large
negative Go favors product formation
S

Keq = [P]/[S]

G=-RTlnKeq

The reaction rate can be increased by lowering

the G (by adding a catalyst).

Rate Acceleration
An enzyme lowers the activation energy,
compared to the uncatalyzed reaction
An enzyme increases the rate of a reaction
(the reaction reaches equilibrium faster), but
does not alter reaction equilibria

Rate Acceleration
A reaction may have several steps, involving
the formation and decay of transient chemical
species known as reaction intermediates
The ES and EP complexes can be considered
reaction intermediates
The ES and EP complexes occupy valleys in
the reaction coordinate diagram
The rate limiting step is the step(s) with the
highest activation energy

How do Es Lower G?
Three distinct but related components:
1) rearrangement of covalent bonds during
catalysis
Amino acid side chains may form a transient covalent bond with
a substrate and activate it for reaction, or a group may be
transiently transferred from the substrate to the enzyme.

How do Es Lower G?
2) Enzymes organize reactive groups into close
proximity
eg of proximity effect, compare the rate of:
Uncatalyzed bimolecular reactions:

two free reactants single restricted transition state

vs

Uncatalyzed unimolecular reactions:

flexible reactant single restricted transition state

vs

Uncatalyzed unimolecular reactions:

inflexible reactant single restricted transition state

How do Es Lower G?
3) Via the energy associated with formation of
noncovalent interactions between the substrate and
the enzyme
Formation of weak, noncovalent interactions (ionic bonds,
H-bonds, Van der Waals, hydrophobic interactions) between
the enzyme and the substrate are used to lower activation
energies.
These interactions are optimized for the transition state (induced
fit model).

Illustration of TS Stabilization:
Imaginary Stickase

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Enzyme Specificity
The same binding energy used for catalysis is also
used to confer specificity to the enzyme.
The E active site is optimized to form a variety of weak
interactions with a particular substrate in the transition
state, thus it will not interact to the same degree with
any other molecule.

What is Enzyme Kinetics?

Kinetics is the study of the rate at which
compounds react

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Why Study Enzyme Kinetics?

Quantitative description of biocatalysis

Determine the order of binding of substrates
Understand catalytic mechanism
Understand regulation of activity
Develop effective inhibitors

How to Do Kinetic Measurements

12

Determination of Kinetic
Parameters
Fit curve to the Michaelis-Menten equation to
calculate parameters Km and Vmax:
v

Vmax [ S ]
Km [S ]

Linearized double-reciprocal plot is old way of

determining the kinetic parameters (still good for
analysis of two-substrate data or inhibition
studies)

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Lineweaver-Burk plot:

Derivation of Enzyme Kinetics

Equations
1. Start with a model mechanism
2. Identify constraints and assumptions
3. Carry out algebra
1) Simplest Model Mechanism: E + S
ES E + P
- One reactant, one product, no inhibitors

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2. Identify Constraints and Assumptions

[E]Tot = [E]f + [ES]
[S]Tot = [S]f + [ES] [S]Tot
Steady state assumption:
d [ ES ]
rate of formation of ES rate of breakdown of ES 0
dt

3. Carry Out the Algebra

The final form in case of a single substrate is

Vmax [ S ]
Km [S ]

or

kcat [ E ]tot [ S ]
Km [S ]

initial velocity (Vo); Vmax maximum velocity

kcat (turnover number): number of substrate

molecules that one enzyme can convert per second
Km (Michaelis constant):

k2 k 1
k1

E+S

k1
k-1

ES

k2

E+P

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Two-substrate Reactions
When two or more reactants are involved, enzyme
kinetics allows you to distinguish between different
kinetic mechanisms (the order of binding of
substrates and release of products)

Sequential mechanism
Ping-Pong mechanism

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Sequential Kinetic Mechanism

A ternary complex (ES1S2) will form

Substrate binding may be random or ordered

Regardless, when a ternary complex forms,
Lineweaver-Burk plots will have intersecting lines

17

Ping-Pong Kinetic Mechanism

18

Enzyme Inhibition
Inhibitors are compounds that decrease enzymes activity
Irreversible inhibitors (inactivators; suicide inhibitors) typically bind to the
enzyme covalently
- one inhibitor molecule can permanently shut off one enzyme molecule
- they are often powerful toxins but also may be used as drugs
Reversible inhibitors bind to, and can dissociate from, the enzyme
- they are often structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme

Reversible inhibitor can bind:

To the free enzyme and prevent the binding of the substrate
To the enzyme-substrate complex and prevent the reaction

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Enzyme Inhibition
Three types of inhibition:
1) competitive
2) uncompetitive
3) noncompetitive (aka mixed)

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Competitive Inhibition
Lines intersect at the y-axis

21

Uncompetitive Inhibition
Lines are parallel

22

Mixed Inhibition
Lines intersect left of the y-axis

Reaction Mechanisms
1. Chymotrypsin

23

 Chymotrypsin is a protease that cleaves

peptides on the C-terminal side of Phe, Tyr,
and Trp residues
Proteases hydrolyze the peptide bond

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25

26

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Reaction Mechanisms
2. enolase

2 Mg+2

28

Reaction Mechanisms
3. Lysozyme

29

Peptidoglycan and Lysozyme

Peptidoglycan is a polysaccharide found in many
bacterial cell walls.
Cleavage of the cell wall by lysozyme leads to the
lysis of bacteria.
Thus, lysozyme is an antibacterial enzyme.

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General Acid-Base + Covalent Catalysis:

Cleavage of Peptidoglycan by Lysozyme
X-ray structures of lysozyme with bound
substrate analogs show that the C-1 carbon is
located between Glu 35 and Asp 52 residues.

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Chapter 6: Summary
In this chapter, we learned about:

why nature needs enzyme catalysis

how enzymes can accelerate chemical reactions
how chymotrypsin breaks down peptide bonds
how to perform and analyze kinetic studies
how to characterize enzyme inhibitors

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