6| Enzymes

CHAPTER 6
Enzymes

Learning goals:
• Physiological significance of enzymes
• Origin of catalytic power of enzymes
• Chemical mechanisms of catalysis
• Mechanisms of chymotrypsin and lysozyme
• Description of enzyme kinetics and inhibition
 What Are Enzymes?
• Enzymes are catalysts.
• increase reaction rates without being used up
• Most enzymes are globular proteins.
• However, some RNA (ribozymes and ribosomal RNA)
also catalyze reactions.
• The study of enzymatic processes is the oldest field of
biochemistry, dating back to late 1700s.
• The study of enzymes has dominated biochemistry in
the past and continues to do so.
Enzyme-Substrate Complex Drives Selectivity
Reaction Coordinate Diagram
Enzymes Decrease ΔG‡
 How to Lower G
Enzymes organize reactive groups into close
proximity and proper orientation.
• Uncatalyzed bimolecular reactions
Two free reactants  single restricted transition state conversion is
entropically unfavorable.
• Uncatalyzed unimolecular reactions
Flexible reactant  rigid transition state conversion is entropically
unfavorable for flexible reactants.
• Catalyzed reactions
The enzyme uses the binding energy of substrates to organize the
reactants to a fairly rigid ES complex.
The entropy cost is paid during binding.
Rigid reactant complex  transition state conversion is entropically
neutral.
 Catalytic Mechanisms
Enzymes may use one or a combination of the
following:
– acid-base catalysis: give and take protons
– covalent catalysis: change reaction paths
– metal ion catalysis: use redox cofactors, pKa
shifters
 Covalent Catalysis
• A transient covalent bond between the enzyme and the
substrate
• Changes the reaction pathway
– uncatalyzed:

– catalyzed (X = catalyst):

• Requires a nucleophile on the enzyme

– can be a reactive serine, thiolate, amine, or carboxylate
 Metal Ion Catalysis

• Involves a metal ion bound to the enzyme

• Interacts with substrate to facilitate binding
– stabilizes negative charges
• Participates in oxidation reactions
 Peptidoglycan and Lysozyme

• Peptidoglycan is a polysaccharide found in many

bacterial cell walls.

• Cleavage of the cell wall leads to the lysis of bacteria.

• Lysozyme is an antibacterial enzyme.

Peptidoglycan and Lysozyme
 What Is Enzyme Kinetics?
• Kinetics is the study of the rate at which compounds
react.
• The rate of enzymatic reaction is affected by:
– enzyme
– substrate
– effectors
– temperature
 Why Study Enzyme Kinetics?

• Quantitative description of biocatalysis

• Determine the order of binding of substrates
• Elucidate acid-base catalysis
• Understand catalytic mechanism
• Find effective inhibitors
• Understand regulation of activity
Derivation of Enzyme Kinetics Equations
1. Start with a model mechanism.
2. Identify constraints and assumptions.
3. Carry out algebra ...
– ... or graph theory for complex reactions.

• Simplest Model Mechanism: E + S  ES  E + P

– one reactant, one product, no inhibitors
Identify Constraints and Assumptions
 
• Total enzyme concentration is constant.
– The mass balance equation for enzymes is ETot = [E] + [ES].
– It is also implicitly assumed that STot = [S] + [ES] ≈ [S].
• Steady state assumption

• What is the observed rate?

– The rate of product formation is the rate of [ES]

breakdown to product.
 Carry Out the Algebra
• The final form in case of a single substrate is the Michaelis-Menten equation:

• kcat (turnover number): how many substrate molecules one enzyme molecule
can convert per second
• Km (Michaelis constant): an approximate measure of a substrate’s affinity for an
enzyme
• During steady state, the maximum velocity (Vmax) occurs when all of the enzyme
is in the ES complex and is dependent on the breakdown of that complex
(k[ES]).
• The microscopic meaning of Km and kcat depends on the details of the
mechanism.
 How to Do Kinetic Measurements
Experiment:
1. Mix enzyme + substrate.
2. Record rate of substrate disappearance and/or product formation as a function of
time (the velocity of reaction).
3. Plot initial velocity versus substrate concentration.
4. Change substrate concentration and repeat.
 Effect of Substrate Concentration

• Ideal rate:

• Deviations due to:

– limitation of measurements
– substrate inhibition
– substrate prep containing inhibitors
– enzyme prep containing inhibitors
Effect of Substrate Concentration
 Saturation Kinetics:
At High [S], Velocity Is not Proportional to [S]
Determination of Kinetic Parameters

A nonlinear Michaelis-Menten plot should be used to

calculate parameters Km and Vmax.

A linearized double-reciprocal plot is good for analysis

of two-substrate data or inhibition.
 Lineweaver-Burk Plot:
Linearized, Double-Reciprocal
 Enzyme Efficiency Is Limited
by Specificity: kcat/KM
• Diffusion from the active site limits the maximum value for
specificity.
• Can gain efficiency by having high velocity or affinity for substrate
– catalase vs. acetylcholinesterase
Enzymes for Which kcat/Km Is Close to the Diffusion-Controlled Limit
TABLE 6-8
(108 to 109 M–1s–1)
kcat/Km
Enzyme Substrate kcat (S–1) Km (S–1)
(M–1s–1)
Acetylcholinesterase Acetylcholine 1.4 x 104 9 x 10–5 1.6 x108
CO2 1 x 106 1.2 x 10–2 8.3 x 107
Carbonic anhydrase
HCO3– 4 x 105 2.6 x 10–2 1.5 x 107
Catalase H2O2 4 x 107 1.1 x 100 4 x 107
Crotonase Crotonyl-CoA 5.7 x103 2 x 10–5 2.8 x 108
Fumarate 8 x 102 5 x 10–6 1.6 x 108
Fumarase
Malate 9 x 102 2.5 x 10–5 3.6 x 107
β-Lactamase Benzylpenicillin 2.0 x 103 2 x 10–5 1 x 108
Source: A. Fersht, Structure and Mechanism in Protein Science, p. 166, W. H. Freeman and Company, 1999.
 Two-Substrate Reactions

• Kinetic mechanism: the order of binding of

substrates and release of products

• When two or more reactants are involved, enzyme

kinetics allows to distinguish between different
kinetic mechanisms:
– sequential mechanism
– ping-pong mechanism
 Sequential Kinetic Mechanism
• We cannot easily distinguish random from ordered.
• Random mechanisms in equilibrium will give the
intersection point at the y-axis.
• Lineweaver-Burk: lines intersect
 Ping-Pong Kinetic Mechanism
Lineweaver-Burk: lines are parallel
 Enzyme Inhibition
Inhibitors are compounds that decrease an enzyme’s
activity.
• Irreversible inhibitors (inactivators) react with the enzyme.
• 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.
• the enzyme-substrate complex and prevent the reaction.
 Competitive Inhibition
• Competes with substrate for binding
– binds active site
– does not affect catalysis

• No change in Vmax; apparent increase in KM

• Lineweaver-Burk: lines intersect at the y-axis.
Competitive Inhibition
 Uncompetitive Inhibition
• Only binds to ES complex
• does not affect substrate binding
• inhibits catalytic function

• Decrease in Vmax; apparent decrease in KM

• No change in KM/Vmax
• Lineweaver-Burk: lines are parallel.
Uncompetitive Inhibition
 Mixed Inhibition

• Binds enzyme with or without substrate

― binds to regulatory site
― inhibits both substrate binding and catalysis
• Decrease in Vmax; apparent change in KM
• Lineweaver-Burk: lines intersect left from the y-axis.
• Noncompetitive inhibitors are mixed inhibitors such that
there is no change in KM.
Mixed Inhibition
 Enzyme Activity Can Be Regulated

• Regulation can be:

– noncovalent modification (allosteric)
– covalent modification
– irreversible
– reversible
 Noncovalent Modification:
Allosteric Regulators
• Allosteric effectors or
modulators are generally
small chemicals.
• Allosteric effectors can
be positive, or improve
enzymatic catalysis.
• Allosteric effectors can
be negative, or reduce
enzymatic catalysis.
 Noncovalent Modification:
Allosteric Regulators
The kinetics of allosteric regulators differ from
Michaelis-Menten kinetics.
Some Reversible Covalent Modifications
 Zymogens Are Activated by
Irreversible Covalent Modification
 Some Enzymes Use
Multiple Types of Regulation
 Chapter 6: Summary

In this chapter, we learned:

• 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
• how enzyme activity can be regulated