Chapter 7

The Behavior of Proteins:

Enzymes, Mechanisms,
and Control

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Chapter Outline
(7-1) Behavior of allosteric enzymes
(7-2) The concerted and sequential models for
allosteric enzymes
(7-3) Control of enzyme activity by phosphorylation
(7-4) Zymogens
(7-5) The nature of the active site
(7-6) Chemical reactions involved in enzyme
mechanisms
(7-7) The active site and transition states
(7-8) Coenzymes

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Allosteric Enzymes
• Allosteric - Term is derived from Greek, which means other
shape
• Allosteric enzyme - Oligomer whose biological activity is affected
by other substances binding to it
• Substances change the enzyme’s activity by altering the
conformation(s) of its 4° structure
• Allosteric effector - Substance that modifies the behavior of an
allosteric enzyme by binding to it
• May be an allosteric inhibitor or allosteric activator
• Aspartate transcarbamoylase (ATCase) - Allosteric protein
involved in feedback inhibition
*An oligomer (oligo-, "a few" + -mer, "parts") is a molecular
complex of chemicals that consists of a few repeating units, in
contrast to a polymer, where the number of monomers is, in
principle, infinite.
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Behavior of Allosteric Enzymes
• ATCase and hemoglobin exhibit cooperative effects caused by
subtle changes in quaternary structure
• Allosteric enzymes have a different response to the presence of
inhibitors from that of non allosteric enzymes
• All enzymes have active sites
• Allosteric enzymes have an active site + allosteric sites
• Non-allosteric enzymes have an active site only.
• Allosteric enzymes like ATCase and myoglobin (the binding of
oxygen to one subunit is affected by it’s interaction with the other
subunits)
• Non allosteric enzymes like myoglobin
• Multimeric proteins (dimers,trimers,tetramers) are usually
allosteric enzymes
• Monomeric proteins are usually non allosteric enzymes
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Feedback Inhibition
• Process by which the final
product of a series of
reactions inhibits the first
reaction in the series

• ATCase enzyme needed

for the reaction between
aspartate and carbamoyl
phosphate to give
carbamoyl aspartate
• How is ATCase inhibited?
by cytidine triphosphate
(CTP) (allosteric inhibitor)
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Figure 7.2 - Schematic Representation of a
Pathway, Showing Feedback Inhibition

Feedback inhibition:
Prevents the
accumulation of unwanted
intermediates by blocking
the whole series.

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Cooperative Behavior of Allosteric Enzymes
• ATCase catalyzes the condensation of aspartate and
carbamoyl phosphate to form carbamoyl aspartate
• Sigmoidal curve describes allosteric behavior
• Hyperbolic curve describes the non-allosteric behavior
• Aspartate is the substrate for which the concentration
is varied
• Concentration of carbamoyl phosphate is kept
constant at high levels

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Figure 7.3 - Enzyme Kinetics of an
Allosteric Enzyme

Cytidine triphosphate (CTP) is an inhibitor

Adenine triphosphate (ATP) is an activator
Control= no inhibitors or activators
With an activator the curve becomes less sigmoidal (closer to hyperbolic curve),
curve shifts to the left.
With an inhibitor the curve becomes more sigmoidal, curve shifts to the right.
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Organization of ATCase
• Catalytic subunits (2 subunits) - Six protein subunits
organized into two trimers (one up and one down)
• Regulatory subunit - Six protein subunits organized
into three dimers
• Catalytic subunits can be separated from
regulatory subunits by treatment with
p-hydroxymercuribenzoate,
which reacts with cysteines in the protein

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Types of Inhibition
• K systems: Enzyme for which the substrate
concentration that yields one-half Vmax is altered by
the presence of inhibitors or activators
• V systems: Enzyme in which the presence of
inhibitor/activator changes the maximal velocity of the
enzyme but not the substrate level that yields one-
half Vmax
• K0.5: Substrate level at one-half Vmax in a K system

• In K systems: Km changes not Vm

• In V systems: Vm changes not Km
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Types of Inhibition (continued)
• Key to allosteric behavior is the existence of multiple
forms for the quaternary structures of allosteric
proteins
• Allosteric effector: Substrate, inhibitor, or activator
that binds to an allosteric enzyme and affects its
activity
• Homotropic effects: Allosteric effects that occur when
several identical molecules are bound to a protein
• Example - Binding of aspartate to ATCase
• Heterotropic effects: Allosteric effects that occur
when different substances are bound to a protein
• Example - Inhibition of ATCase by cytidine triphosphate
(CTP) and activation by adenosine triphosphate (ATP)
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Concerted Model
• Proposed by Monod, Wyman, and Changeux in1965
• Description of allosteric activity in which the
conformations of all subunits change simultaneously
• Protein has two conformations
• Active R (relaxed) - Binds substrate tightly
• Inactive T (tight or taut) - Binds substrate less tightly
• In the absence of substrate, most enzyme molecules
are in the T (inactive) form
• Presence of substrate shifts the equilibrium from the T
(inactive) form to the R (active) form
• In changing from T to R and vice versa, all subunits
change conformation simultaneously
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Figure 7.6 - Monod–Wyman–Changeux (MWC) Model for
Allosteric Transitions, Also Called the Concerted Model

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Concerted Model (continued)
• Explains the sigmoidal effects
• Higher L means higher favorability of free T form
• Lower c means higher affinity between S and R form
and more sigmoidal effects

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Figure 7.8 - Effects of Binding Activators
and Inhibitors with the Concerted Model

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Sequential Model
• Binding of substrate induces a conformational
change from the T form to the R form
• Cooperative binding of substrate to an allosteric
enzyme
• R form is favored by allosteric activator
• Allosteric inhibition occurs by the induced-fit
mechanism
• Unique feature
• Negative cooperativity: Cooperative effect whereby
binding of the first ligand to an enzyme or protein
causes the affinity for the next ligand to be lower

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Figure 7.9 - Sequential Model of Cooperative
Binding

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Control of Enzyme Activity by
Phosphorylation
• Side-chain hydroxyl groups of serine, threonine, and
tyrosine can form phosphate esters
• Phosphorylation by ATP can convert an inactive
precursor into an active enzyme
• Example - Transport across membranes

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Phosphorus
•  15P: [Ne] 3s2 3p3

• because it is highly reactive, P occurs most

commonly in nature as phosphate rocks, which are
mostly calcium phosphate.
• Phosphates (compounds containing the phosphate
ion, PO43−) are a component of DNA, RNA, ATP, and
phospholipids.
• White P consists of discrete tetrahedral P4 molecules

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Figure 7.11 - Phosphorylation of Serine,
Threonine, and Tyrosine

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Figure 7.12 - Phosphorylation of the Sodium–Potassium Pump Is
Involved in Cycling the Membrane Protein between the Form That
Binds to Sodium and the Form That Binds to Potassium

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Figure 7.13 - Glycogen Phosphorylase Activity Is Subject to
Allosteric Control and Covalent Modification via
Phosphorylation

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Zymogen
• Inactive protein that can be activated by specific
hydrolysis of peptide bonds
• Chymotrypsinogen
• Synthesized and stored in the pancreas
• Consists of a single polypeptide chain of 245 amino
acid residues cross-linked by five disulfide (—S—S—)
bonds
• When secreted into the small intestine, the digestive
enzyme trypsin cleaves a polypeptide bond from the
N-terminal end to give active -chymotrypsin

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Figure 7.15 - The Proteolytic Activation of
Chymotrypsinogen

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Chymotrypsin
• 15-unit polypeptide remains bound to -chymotrypsin
by a single disulfide bond
• -chymotrypsin catalyzes the hydrolysis of two
dipeptide fragments to give -chymotrypsin
• -chymotrypsin consists of three polypeptide chains
joined by two of the five original disulfide bonds
• Changes in 1° structure that accompany the change
from chymotrypsinogen to -chymotrypsin result in
changes in 3° structure
• -chymotrypsin is enzymatically active because of its
3° structure, just as chymotrypsinogen was inactive
because of its 3° structure
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Nature of the Active Site
• Important questions to ask about mode of action of
an enzyme
• Which amino acid residues on an enzyme are in the
active site and catalyze the reaction?
• What is the spatial relationship of the essential amino
acids residues in the active site?
• What is the mechanism by which the essential amino
acid residues catalyze the reaction?

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Nature of the Active Site: Chymotrypsin
• Enzyme of the digestive system that catalyzes:
• Hydrolysis of peptide bonds adjacent to aromatic
amino acid residues in the protein being hydrolyzed
• Hydrolysis of esters in model studies in laboratories

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Kinetics of Chymotrypsin Reaction
• p-nitrophenyl acetate is
hydrolyzed by
chymotrypsin in two
stages
• At the end of stage one,
the p-nitrophenolate ion
is released
• At stage two, acyl-
enzyme intermediate is
hydrolyzed, releasing
acetate and regenerating
free enzyme

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Figure 7.16 - Chymotrypsin-Catalyzed
Reaction of p-Nitrophenyl Acetate

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Determining the Essential Amino Acids in
the Active Site
• Chymotrypsin is a serine protease
• Serine proteases: Class of proteolytic enzymes in
which a serine hydroxyl plays an essential role in
catalysis
• Diisopropylphosphofluoridate (DIPF) inactivates
chymotrypsin by reacting with serine 195, and verifies
that the residue is at the active site

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Determining the Essential Amino Acids in
the Active Site (continued 1)
• Histidine 57 is critical for
activation of
chymotrypsin
• Can be chemically
labeled by N-tosylamido-
L-phenylethyl
chloromethyl ketone
(TPCK)

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Determining the Essential Amino Acids in
the Active Site (continued 2)
• Serine 195 and histidine 57 are required for the
activity of chymotrypsin
• Must be close to each other in the active site
• Aspartate 102 is involved in catalysis at the active
site
• Results of X-ray crystallography show the definite
spatial relationship among active site-residues
• Folding of the chymotrypsin backbone, mostly in an
antiparallel pleated sheet array, positions the essential
amino acid residues around the active-site pocket

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Figure 7.23 - Three-Dimensional Structure
of the Active Site

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Mechanism of Catalysis
• Oxygen of the serine side chain is a
nucleophile
• Nucleophile: Electron-rich substance that
tends to react with sites of positive charge
or polarization
• Attacks carbonyl group of peptide group

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Catalytic Mechanisms
• Nucleophilic substitution reactions: One functional
group is replaced by another as the result of
nucleophilic attack
• SN1
• Unimolecular nucleophilic substitution
• Rate of reaction follows first-order kinetics
• SN2
• Bimolecular nucleophilic substitution
• Rate of reaction follows second-order kinetics
• General acid–base catalysis: Depends on transfer
of protons

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Metal–Ion Catalysis
• Called Lewis acid–base catalysis
• Depends on the Lewis definition of an acid as an
electron-pair acceptor and a base as an electron-pair
donor
• Lewis acids - Mn2+, Mg2+, and Zn2+
• Essential components of many enzymes
• Carboxypeptidase A requires Zn2+ for enzyme activity

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Metal–Ion Catalysis (continued)
• Zn2+ of carboxypeptidase A
• Complexed to the imidazole side chains of histidines
69 and 196 and to the carboxylate side chain of
glutamate 72
• Activates the carbonyl group
for nucleophilic acyl
substitution

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Specificity of Enzymes
• Absolute specificity - Catalyzes the reaction of one
unique substrate to a particular product
• Relative specificity - Catalyzes the reaction of
structurally related substrates to give structurally
related products
• Stereospecificity - Catalyzes a reaction in which one
stereoisomer is reacted or formed in preference to all
others that might react or form
• Example - Hydration of a cis alkene (but not its trans
isomer) to give an R alcohol (but not the S alcohol)

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Figure 7.28 - Asymmetric Binding Site on an Enzyme Can
Distinguish between Identical Groups, Such as A and B

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Nature of Transition States
• Enzyme provides an alternative pathway with a lower
activation energy
• True nature of transition state is a chemical species
that is intermediate in structure between the
substrate and the product
• Transition state has a different shape from either the
substrate or the product
• Transition-state analog: Synthesized compounds
that mimic the form of the transition state of an
enzyme reaction

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Nature of Transition States (continued)
• In 1969, Jencks proposed that an immunogen would
elicit antibodies with catalytic activity if the
immunogen mimicked the transition state of the
reaction
• Lerner and Schultz created the first catalytic antibody,
or abzyme, in 1986
• Abzymes: Antibodies that are produced against a
transition-state analog and that have catalytic activity
similar to that of a naturally occurring enzyme

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Figure 7.29 - Proline Racemase
Reaction

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Coenzymes
• Nonprotein substances that take part in enzymatic
reactions and are regenerated at the end of the
reaction
• Metal ions can behave as coordination compounds
• Zn2+ and Fe2+
• Organic coenzymes - Vitamins and their derivatives
• Involved in oxidation–reduction reactions
• Serve as group-transfer agents in metabolic processes

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Nicotinamide Adenine Dinucleotide (NAD+)
• Coenzyme in many
oxidation–reduction
reactions
• Structure - Consists of a
nicotinamide ring, an
adenine ring, and two
sugar-phosphate groups
linked together

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Nicotinamide Adenine Dinucleotide (NAD+)
(continued)

• Two-electron oxidizing agent

• Reduced to NADH
• Nicotinamide ring is where reduction-oxidation occurs

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B6 Vitamins
• Pyridoxal, pyridoxamine, and pyridoxine and their
phosphorylated forms
• Coenzymes involved in amino group transfer from
one molecule to another
• Important in biosynthesis of amino acids

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B6 Vitamins (continued)
• Pyridoxal and pyridoxamine phosphates are involved
in the transfer of amino groups in a reaction called
transamination

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