Mary K.

Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell

Chapter Seven
The Behavior of Proteins:
Enzymes, Mechanisms, and Control

Paul D. Adams University of Arkansas

Allosteric Enzymes
Allosteric: Greek allo + steric, other shape
Allosteric enzyme: an oligomer whose biological activity is affected by
other substances binding to it

these substances change the enzymes activity by altering the

conformation(s) of its 4structure
Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an

allosteric inhibitor
allosteric activator
Aspartate transcarbamoylase (ATCase)

feedback inhibition

Feedback Inhibition
Formation of product
inhibits its continued
production

ATCase
Rate of ATCase catalysis vs
substrate concentration

Sigmoidal shape of curve describes

allosteric behavior
ATCase catalysis in presence
of CTP; ATP

ATCase (Contd)
Organization of ATCase
catalytic unit: 6 subunits
organized into 2 trimers
regulatory unit: 6 subunits
organized into 3 dimers
Catalytic subunits can be
separated from regulatory
subunits by a compound that
reacts with cysteine (phydroxymercuribenzoate)

Allosteric Enzymes (Contd)

Two types of allosteric enzyme systems exist

Note: for an allosteric enzyme, the substrate

concentration at one-half Vmax is called the K0.5
K system: an enzyme for which an inhibitor or
activators alters K0.5
V system: an enzyme for which an inhibitor or
activator alters Vmax but not K0.5

Allosteric Enzymes (Contd)

The key to allosteric behavior is the existence of multiple
forms for the 4structure of the enzyme

allosteric effector: a substance that modifies the 4

structure of an allosteric enzyme
homotropic effects: allosteric interactions that occur
when several identical molecules are bound to the
protein; e.g., the binding of aspartate to ATCase
heterotropic effects: allosteric interactions that occur
when different substances are bound to the protein;
e.g., inhibition of ATCase by CTP and activation by
ATP

The Concerted Model

Wyman, Monod, and Changeux - 1965
The enzyme has two conformations
R (relaxed): binds substrate tightly; the active form
T (tight or taut): binds substrate less tightly; the
inactive form
in the absence of substrate, most enzyme molecules
are in the T (inactive) form
the 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; all changes are
concerted

Concerted Model (Contd)

A model represented by a protein having two conformations
Active (R) form-Relaxed binds substrate tightly, Inactive (T) formTight (taut) binds substrate less tightly both change from T to R at
the same time
Also called the concerted model
Substrate binding shifts equilib. To the relaxed state.
Any unbound R is removed KR<KT
Ratio of dissociation constants is called c
The Monod-Wyman-Changeaux
model

Concerted Model (Contd)

The model explains the sigmoidal effects
Higher L means higher favorability of free T form
Higher c means higher affinity between S and R form,
more sigmoidal as well.

Concerted Model (Contd)

An allosteric activator (A) binds to and stabilizes the R
(active) form
An allosteric inhibitor (I) binds to and stabilizes the T
(inactive) form
Effect of
binding
activators
and inhibitors

Sequential Model (Contd)

Main Feature of Model:

the binding of substrate induces a conformational

change from the T form to the R form
the change in conformation is induced by the fit of the
substrate to the enzyme, as per the induced-fit model
of substrate binding

sequential model represents cooperativity

Sequential Model (Contd)

Sequential model for cooperative binding of substrate to an allosteric enzyme
R form is favored by allosteric activator
Allosteric inhibition also occurs by the induced-fit mechanism
Unique feature of Sequential Model of behavior:
Negative cooperativity- Induced conformational changes that make the enzyme
less likely to bind more molecules of the same type.
Sequential Model:

Control of Enzyme Activity via Phosphorylation

The side chain -OH groups
of Ser, Thr, and Tyr can
form phosphate esters
Phosphorylation by ATP can
convert an inactive
precursor into an active
enzyme

Membrane transport is a
common example

Membrane Transport
Source of PO4 is ATP
When ATP is hydrolyzed, energy released that allows other
energetically unfavorable reactions to take place
PO4 is donated to residue in protein by protein kinases

Zymogens
Zymogen: Inactive precursor of an enzyme where cleavage
of one or more covalent bonds transforms it into the active
enzyme
Chymotrypsinogen
synthesized and stored in the pancreas
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 15 unit polypeptide from the Nterminal end to give -chymotrypsin

Activation of chymotrypsin
Activation of chymotrypsinogen by proteolysis

Chymotrypsin
A15-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 1structure that accompany the change from
chymotrypsinogen to -chymotrypsin result in changes in
2- and 3structure as well.
-chymotrypsin is enzymatically active because of its 2and 3structure, just as chymotrypsinogen was inactive
because of its 2- and 3structure

The Active Site

Some important questions to ask about enzyme mode of action:
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?

As a model, we consider chymotrypsin, an enzyme of the

digestive system that catalyzes the selective hydrolysis of
peptide bonds in which the carboxyl group is contributed by
Phe or Tyr

Kinetics of Chymotrypsin Reaction

p-nitrophenyl acetate is
hydrolyzed by
chymotrypsin in 2
stages.
At the end of stage 1,
the p-nitrophenolate ion
is released.
At stage 2, acyl-enzyme
intermediate is
hydrolyzed and acetate
(Product) is
releasedfree enzyme
is regenerated

Chymotrypsin
Reaction with a model substrate

Chymotrypsin (Contd)
Chymotrypsin is a serine protease

DIPF inactivates chymotrypsin by reacting with

serine-195, verifying that this residue is at the active
site

Chymotrypsin (Contd)
H57 also critical for
activation of enzyme
Can be chemically
labeled by TPCK

Chymotrypsin (Contd)
Because Ser-195 and His-57 are required for activity,
they must be close to each other in the active site
Results of x-ray crystallography show the definite
arrangement of amino acids at the active site
In addition to His-57 and Ser-195, Asp-102 is also
involved in catalysis at the active site
The folding of the chymotrypsin backbone, mostly in
antiparallel pleated sheet array, positions the essential amino
acids around the active-site pocket

Chymotrypsin (Contd)
The active site of
chymotrypsin shows
proximity of 2 reactive
a.a.

Mechanism of Action of Critical Amino Acids in

Chymotrypsin
Serine oxygen is nucleophile
Attacks carbonyl group of peptide bond

Catalytic Mechanisms
General acid-base catalysis: depends on donation
and acceptance of protons (proton transfer reactions)
Nucleophilic substitution catalysts- Nucleophilic
electron-rich atom attacks electron deficient atom.
same type of chemistry can occur at active site of
enzyme: SN1, SN2

Catalytic Mechanisms (Contd)

Lewis acid/base reactions
Lewis acid: an electron pair acceptor
Lewis base: an electron pair donor
Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential
components of many enzymes (metal ion catalysts)
carboxypeptidase A requires Zn2+ for activity

Catalytic Mechanisms (Contd)

Zn2+ of
carboxypeptidase is
complexed with:
The imidazole side
chains of His-69 and
His-196 and the
carboxylate side
chain of Glu-72
Activates the
carbonyl group for
nucleophilic acyl
substitution

Enzyme Specificity
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 be reacted or formed

example: hydration of a cis alkene (but not its trans

isomer) to give an R alcohol (but not the S alcohol)

Asymmetric binding
Enzymes can be
stereospecific
(Specificity where
optical activity may pay
a role)
Binding sites on enzymes
must be asymmetric

Active Sites and Transition States

Enzyme catalysis

an enzyme provides an alternative pathway with a lower

activation energy
the transition state often has a different shape than either the
substrate(s) or the product(s)
True nature of transition state is a chemical species that is
intermediate in structure between the substrate and the product.

Transition state analog: a substance whose shape mimics that of a

transition state
In 1969 Jenks proposed that

an immunogen would elicit an antibody with catalytic activity if

the immunogen mimicked the transition state of the reaction
the first catalytic antibody or abzyme was created in 1986 by
Lerner and Schultz

*(Biochemical Connections, p. 196)

Coenzymes
Coenzyme: a nonprotein substance that takes part in an
enzymatic reaction and is regenerated for further reaction
metal ions- can behave as coordination compounds. (Zn2+,
Fe2+)
organic compounds, many of which are vitamins or are
metabolically related to vitamins (Table 7.1).

NAD+/NADH
Nicotinamide adenine
dinucleotide (NAD+) is used
in many redox reactions in
biology.
Contains:
1) nicotinamide ring
2) Adenine ring
3) 2 sugar-phosphate groups

NAD+/NADH (Contd)
NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
Nicotinamide ring is where reduction-oxidation
occurs

B6 Vitamins
The B6 vitamins are coenzymes involved in amino group
transfer from one molecule to another.
Important in amino acid biosynthesis

Pyridoxal Phosphate
Pyridoxal and pyridoxamine phosphates are involved in
the transfer of amino groups in a reaction called
transamination

Figure 7.21 p. 197