6| Enzymes

© 2017 W. H. Freeman and Company

 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.
Why Biocatalysis Over Inorganic Catalysts?
• Greater reaction specificity: avoids side products
• Milder reaction conditions: conducive to conditions in cells
pH ~ 7, 37°C
• Higher reaction rates: in a biologically useful timeframe
• Capacity for regulation: control of biological pathways

- -
COO COO
NH2 • Metabolites have many
- potential pathways of
O COO
OH COO
- decomposition.

-
-
OH
O COO
Chorismate
COO
- • Enzymes make the
COO mutase -
OOC
O
desired one most
favorable.
NH2 OH
 Enzymatic Substrate Selectivity
OH
H
H
- +
OOC NH3 - +
OOC NH3

H
-
OOC
+
NH3 No binding
OH

HO OH
H

H
H Binding but no reaction
NH
CH3

Example: phenylalanine hydroxylase

Enzyme-Substrate Complex Drives Selectivity
Reaction Coordinate Diagram
Enzymes Decrease ΔG‡
 How to Lower G

Enzymes bind transition states best.

• The idea was proposed by Linus Pauling in 1946.
– Enzyme active sites are complimentary to the
transition state of the reaction.
– Enzymes bind transition states better than
substrates.
– Stronger/additional interactions with the transition
state as compared with the ground state lower the
activation barrier.
Largely ΔH‡ effect
Illustration of TS Stabilization Idea:
Imaginary Stickase
 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
 Chymotrypsin
• During digestion, dietary proteins must be broken down into
small peptides by proteases.
• Chymotrypsin is one of several proteases that cuts peptides
at specific locations on the peptide backbone.
• This protease is able to cleave the peptide bond adjacent to
aromatic amino acids.
Chymotrypsin cuts this bond.
Chymotrypsin Uses Most of
the Enzymatic Mechanisms
Chymotrypsin Mechanism
Step 1: Substrate Binding
Chymotrypsin Mechanism
Step 2: Nucleophilic Attack
Chymotrypsin Mechanism
Step 3: Substrate Cleavage
Chymotrypsin Mechanism
Step 4: Water Comes In
Chymotrypsin Mechanism
Step 5: Water Attacks
Chymotrypsin Mechanism
Step 6: Break-off from the Enzyme
Chymotrypsin Mechanism
Step 7: Product Dissociates
 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
 Carry Out the Algebra
• The final form in case of a single substrate is the Michaelis-Menten equation:

k cat [E tot ][S] V max[S]

v 
K m  [S] Km  [S]
• 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
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 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
 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