Chapter 7.

Enzyme Mechanisms
▪ 7.1 Overview of Enzymes
▪ 7.2 Enzyme Structure and Function
▪ 7.3 Enzyme Reaction Mechanisms
▪ 7.4 Enzyme Kinetics
▪ 7.5 Regulation of Enzyme Activity
7.1 Overview of Enzymes
▪ 1926 – James Sumner discovered that enzymes are proteins.
▪ Urease was the first enzyme to be crystallized and purified.
▪ 1929 – John Northrop purified pepsin.

▪ Until 1958, biochemists thought that the observed high

specificity of enzyme-mediated catalysis was best explained by
rigid physical and chemical complementarity between the
reactant, usually referred to as the substrate, and the enzyme.
James Sumner provided the first evidence that a protein can function as an enzyme when he
purified and crystallized the enzyme urease from jack bean protein extracts. Crystallization of
the protein was an important step to demonstrate its purity. a. James Sumner (1887–1955) was
a biochemist at Cornell University when he first purified urease in 1926. b. Jack beans
(Canavalia ensiformis) contain high levels of the enzyme urease. c. Protein crystals of purified
urease isolated from jack beans.

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Enzyme Specificity
▪ Lock and key
• Substrate (reactant) binds to the enzyme perfectly.
▪ Induced fit
• Enzyme is flexible to accommodate the ill-fitting substrate.
• Permits a much larger number of weaker interactions between
the substrate and enzyme
Hexokinase: An Induced Fit Mechanism
Conformational Selection Mechanism
▪ Suggests that all conformations preexist, although ligand binding
shifts the preference for a particular conformation
▪ Once a primary conformation is “trapped” by ligand binding,
induced fit may optimize the interactions.
▪ Interactions between the enzyme and substrate promote
catalysis.
Critical Aspects of Enzyme Structure and Function, Part 1
1. Enzymes usually bind to
substrates with high affinity
and specificity.
• Active sites (binding
pockets) in the enzyme
bind to the substrate and
promote catalytic reactions.
Critical Aspects of Enzyme Structure and Function, Part 2
2. Substrate binding to the active site induces changes in the
enzyme.
• Example: hexokinase, a metabolic enzyme
Critical Aspects of Enzyme Structure and Function, Part 3
3. Enzyme activity is
highly regulated in
cells.
▪ Modes of enzyme
regulation:
• Bioavailability
• Catalytic efficiency

The catalytic efficiency of glycogen phosphorylase is increased by noncovalent binding of allosteric regulators, such as
AMP, and by covalent attachment of a phosphoryl group to Ser14. The bound coenzyme pyridoxal phosphate is shown in
the active site. In glycogen phosphorylase, the regulatory sites are not directly at the active site. However, the binding of
AMP or serine phosphorylation at the regulatory sites causes conformational changes that affect the catalytic efficiency in
the active site.
Enzymes Are Chemical Catalysts
▪ Alter the rates of reaction without changing the ratio of
substrates and products at equilibrium
▪ Decrease the activation energy to speed up a reaction
Reaction Coordinate Diagram
Cofactors and Coenzymes
▪ Cofactors
• Small molecules that aid in the catalytic reaction within the
active site
• Include inorganic ions such as Fe2+, Cu2+, and Mg2+
▪ Coenzyme
• Enzyme cofactors that require organic components
• Include vitamin-derived species such as NAD+ and FAD
▪ Prosthetic groups – coenzymes that are permanently associated
with enzymes
Common Cofactors Used in Catalysis
Nitrite reductase is an enzyme present in several types of soil bacteria that recycle nitrogen
in the environment. As seen here in the molecular structure of the enzyme’s active site, the
Cu2+ ion is coordinated to two histidine residues that function to hold the metal cofactor in an
optimal position for catalyzing the reduction of the substrate nitrite (NO2−) to form the product,
nitric oxide.

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Coenzyme Vitamin source Representative enzyme Role in catalysis
Nicotinamide adeniamide Vitamin B subscript 3 (niacin) Lactate dehydrogenase Oxidation-reduction
adenine dinucleotide (positive
1 N A D/N A D H)
Flavin adenine dinculeotide (F Vitamin B subscript 2 Succinate dehydrogenase Oxidation-reduction
A D/F A D H subscript 2)
Thiamine pyrophosphate (T P Vitamin B subscript 1 Pyruvate dehydrogenase Aldehyde group transfer
P)
Biotin Vitamin C subscript 7 Pyruvate carboxylase Carbonxylation
Coenzyme A (C o A) Pantothenic acid Acetyl-CoA carboxylase Acyl group transfer
Tetrahydrofolate (T H F) Folate Thymidykate synthase Single carbon transfer
Pyridoxal phosphate (P L P) Vitamin B subscript 6 Aspartate aminotransferase Amine group transfer
Lipoamide Lipoic acid Pyruvate dehydrogenase Two-carbon transfer
Cobalamin Vitamin B subscript 12 Methylmalonyl- CoA mutase Alkyl group transfer

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NAD+/NADH Redox Reaction

Nicotinamide adenine dinucleotide is a coenzyme in the lactate dehydrogenase reaction. a. Chemical structure of
nicotinamide adenine dinucleotide in the oxidized (NAD+) and reduced (NADH) forms. b. Molecular structure of
part of the lactate dehydrogenase enzyme from the organism Plasmodium falciparum, with an NADH molecule
bound to the active site.
Lipoamide as a Coenzyme

Lipoamide is a covalently attached coenzyme that plays a key role in several decarboxylase reactions. a. A lipoyl
group is attached to the amino group of lysine residues in enzymes, shown here in both the oxidized (lipoamide)
and reduced (dihydrolipoamide) forms. b. The pea glycine carboxylase enzyme contains a lipoamide coenzyme
that protrudes into the enzyme active site. This protein structure contains the reduced form of lipoamide, called
dihydrolipoamide.
Enzyme Nomenclature
▪ Most enzymes end in “-ase.”
▪ The substrate is usually included in the name.

Number Enzyme class Type of reaction Generic enzymes

1 Oxidoreductase Oxidation reduction, transfer of H or Oxidase, dehydrogenases

O atoms
2 Transferase Transfer of functional groups, Kinases, transaminases
example methyl, acyl, amino,
phosphoryl
3 Hydrolase Formation of two products by Peptidases, lipases
hydrolyzing a substrate
4 Lyase Cleavage of C C, C O, C N, and other Decarboxylases, carboxylases
bonds by means other than
hydrolysis or oxidation
5 Isomerase Intramolecular rearrangements, Mutases, isomerases
transfer of groups within molcules
6 Ligase Formation of C C, C O, C S, or C N Synthetases
bonds using A T P cleavage
7.2 Enzyme Structure and Function
▪ Enzymes increase the rate of reaction in three major ways:
• They lower the activation energy by stabilizing the transition
state.
• They provide an alternate path for product formation.
• They reduce entropy by orienting the substrates appropriately
for the reaction to occur.
Physical and Chemical Properties of Active Sites
Active Sites Contribute to Catalytic Properties
1. Sequestered microenvironment of the active site
• Provides optimal orientation of the substrate relative to the
reactive chemical group
• Excludes excess solvent
2. Binding interactions between the substrate and the enzyme to
create a transition state
3. Presence of catalytic functional groups
 Biochemistry, First Edition
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The enzyme active site of hexokinase excludes H2O molecules upon glucose binding, thereby
preventing nonproductive phosphoryl transfer from ATP to H2O. a. In the absence of glucose,
H2O molecules (shown as red spheres) occupy the solventexposed active site. BASED ON
PDB FILE 1IG8. b. A conformational change is induced upon glucose binding at the active site.
Glucose replaces water in the active site by occupying a similar volume and using similar weak
interactions for binding. Therefore, the H2O molecules are expelled from the active site.

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Transition State Analogs
▪ Stable molecules that mimic the proposed transition state
▪ These molecules bind tightly to the active site.
Catalytic Functional Groups

Amino acid General acid form General base form

His Chemical structure of imidazole ring shows a five- Chemical structure of imidazole
member ring with nitrogen atom occupying ring shows a five-member ring
positions at first and third. A double bond exists with nitrogen atom occupying
between carbon at second and nitrogen at third positions at first and third. A
positions and between carbon at fourth and fifth double bond exists between
positions. The nitrogen atom at first position is carbon at second and nitrogen at
bonded to a hydrogen atom. The carbon atom at third positions and between
fourth position is bonded to an R group. The carbon at fourth and fifth
nitrogen atom at third position is bonded to a positions. The nitrogen atom at
hydrogen atom and carries a positive charge. The first position is bonded to a
hydrogen atom is highlighted. hydrogen atom. The carbon atom
at fourth position is bonded to an
R group.
A s p, G l u R group is bonded to a carboxylic acid group. The R group is bonded to a
hydrogen atom of the carboxylic acid group is carboxylate anion.
highlighted.
S e r, T h r R group is bonded to a hydroxyl group. The R group is bonded to an oxygen
hydrogen atom is highlighted. anion.

Tyr R group is bonded to a benzene ring. At para R group is bonded to a benzene

position, the benzene ring is further bonded to a ring. At para position, the
hydroxyl group. benzene ring is further bonded to
an oxygen anion.
Cys R group is bonded to a thiol group. The hydrogen R group is bonded to a sulfur
atom is highlighted. anion.

Lys R group is bonded to a nitrogen ion carrying R group is bonded to an amine.

positive charge, which is further bonded to three
hydrogen atoms. The hydrogen atom is
highlighted.
Arg R group is bonded to a nitrogen atom, which is R group is bonded to a nitrogen
further bonded to a hydrogen atom and a carbon atom, which is further bonded to
atom. The carbon atom is further bonded to a a hydrogen atom and a carbon
nitrogen ion carrying positive charge by a double atom. The carbon atom is further
bond and an amine group. The nitrogen ion bonded to a nitrogen atom by a
carrying positive charge is bonded to two double bond and an amine group.
hydrogen atoms. The hydrogen atom is The nitrogen is further bonded to
highlighted. a hydrogen atom.
Common Catalytic Mechanisms, Part 1
1. Acid–base catalysis
• Proton transfer
that either
involves water
(specific acid–
base) or a
functional group
(general acid–
base)
Common Catalytic Mechanisms, Part 2
2. Covalent catalysis
• Nucleophile group
on the enzyme
attacks an
electrophile
center on the
substrate to form
a covalent
enzyme–substrate
intermediate.
Common Catalytic Mechanisms, Part 3
3. Metal ion catalysis
• Metals are used to promote
proper orientation of
bound substrates and can
aid in redox reactions.
Enzyme-Mediated Reactions, Part 1
1. Coenzyme-dependent redox reactions
• Include dehydrogenases
• Involve NAD+/NADH, NADP+/NADPH, FAD/FADH2, and
FMN/FMNH2
Enzyme-Mediated Reactions, Part 2
2. Metabolic
transformation
reactions
• Involve
isomerizations,
condensations, and
dehydration
(hydrolysis) reactions
Enzyme-Mediated Reactions, Part 3
3. Reversible covalent modifications
• Act as molecular switches by turning on/off cell signaling and
gene expression
• Include kinases and phosphatases
• ATP is commonly used as a phosphoryl group source.
7.3 Enzyme Reaction Mechanisms
▪ Substrates bind to enzyme active sites through weak noncovalent
interactions.
▪ Enzymes use conventional catalytic reaction mechanisms that
follow basic principles of organic chemistry.
Bovine chymotrypsin consists of three polypeptide chains linked together by disulfide bonds. a. Ribbon structure of
chymotrypsin, highlighting the two interstrand and three intrastrand disulfide bonds. It also shows the location of the
enzyme active site, indicated by the space-filling model of a small chymotrypsin inhibitor. One of the disulfide bonds is
behind the inhibitor and is not visible in this orientation. b. Map of the A, B, and C chains of chymotrypsin, illustrating the
disulfide bonds and the catalytic triad residues in the active site.

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Chymotrypsin: A Serine Protease
▪ Involves covalent and acid–base catalysis
▪ Uses a catalytic triad (His, Asp, Ser) to form a hydrogen-bonded
network required for catalysis
▪ Ser is converted to a highly reactive nucleophile.
Catalytic Mechanism for Chymotrypsin
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Enzyme Specificity for Binding Pockets
Enolase: A Metalloenzyme
▪ Glycolytic enzyme that is also used in gluconeogenesis
▪ Active site contains two divalent metal ions required for catalysis
Enolase’s Active Site
Catalytic Mechanism for Enolase
HMG-CoA Reductase
▪ An enzyme involved in cholesterol biosynthesis
▪ Inhibitors directly reduce serum cholesterol levels.
▪ Contains four active sites for the substrate and cofactor (NADPH)
to bind
HMG-CoA Reductase Mechanism