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Chapter Outline
7-1 The 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
Signals regulate the flow of traffic
in much the same fashion as control
mechanisms in chemical reactions
7-7 The Active Site and Transition States
7-8 Coenzymes
Fig. 7-CO, p. 165
Allosteric Enzymes
Feedback Inhibition
• Allosteric: Greek allo + steric, other shape
• Allosteric enzyme: an oligomer whose biological activity is affected by
other substances binding to it
Formation of product inhibits its continued production
• these substances change the enzyme’s activity by altering the
conformation(s) of its 4°structure
• Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an
• allosteric inhibitor
• allosteric activator
• Aspartate transcarbamoylase (ATCase)
• feedback inhibition
• How are Allosteric Enzymes Controlled?
• Allosteric enzymes can be controlled by many
different mechanisms, including inhibition and
activation by reversibly-binding molecules.
• Feedback inhibition is a common way to regulate an
allosteric enzyme that is part of a complicated
pathway.
p. 166
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ATCase
• What molecule acts as a positive effector (activator)
of ATCase? What molecule acts as an inhibitor?
• Rate of ATCase catalysis vs substrate concentration
• ATP acts as a positive effector
• ATCase catalysis in presence
of CTP; ATP
• Sigmoidal shape of curve describes allosteric behavior
• CTP acts as an inhibitor
• What features distinguish enzymes that undergo
allosteric control from those that obey the MichaelisMenten equation?
• How is the cooperative behavior of allosteric
enzymes reflected in a plot of reaction rate against
substrate concentration?
• Allosteric enzymes display sigmoidal kinetics when rates are
plotted versus substrate concentration.
• Enzymes that exhibit cooperativity do not show
hyperbolic curves of rate versus substrate
concentration. Their curves are sigmoidal. The level
of cooperativity can be seen by the shape of the
sigmoidal curve.
• Michaelis–Menten enzymes exhibit hyperbolic kinetics.
• Allosteric enzymes usually have multiple subunits, and the
binding of substrates or effector molecules to one subunit
changes the binding behavior of the other subunits.
• Does the behavior of allosteric enzymes become
more or less cooperative in the presence of
activators?
• Activators make the shape of the curve less
sigmoidal.
• Does the behavior of allosteric enzymes become
more or less cooperative in the presence of
inhibitors?
• Inhibitors make the shape of the curve more
sigmoidal.
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ATCase (Cont’d)
Allosteric Enzymes (Cont’d)
• Organization of ATCase
• catalytic unit: 6 subunits
organized into 2 trimers
• regulatory unit: 6 subunits
organized into 3 dimers
• Two types of allosteric enzyme systems exist
• Catalytic subunits can be
separated from regulatory
subunits by a compound that
reacts with cysteine (phydroxymercuribenzoate)
Allosteric Enzymes (Cont’d)
• The key to allosteric behavior is the existence of multiple
forms for the 4°structure 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
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
Summary
• Allosteric enzymes exhibit different behaviors
compared to non-allosteric enzymes, and the
Michaelis-Menten equations are not applicable.
• A plot of velocity vs. [S] for an allosteric enzyme has
a sigmoidal shape.
• One type of control often seen with allosteric
enzymes is called feedback inhibition.
• Inhibitors and activators can control the activity of an
allosteric enzyme.
The Concerted Model
Concerted Model (Cont’d)
• 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
• 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
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Concerted Model (Cont’d)
• The model explains the sigmoidal effects
• Higher L means higher favorability of free T form
• Lower c means higher affinity between S and R form,
more sigmoidal as well.
Concerted Model (Cont’d)
• 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 (Cont’d)
Sequential Model (Cont’d)
• Main Feature of Model:
Sequential model for cooperative binding of substrate to an allosteric enzyme
• 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
• 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:
• sequential model represents cooperativity
Summary
• What is the Sequential Model for Allosteric Behavior?
• In the sequential model, the binding of substrate
induces the conformational change in one subunit,
and the change is subsequently passed along to
other subunits.
• The two principal models for allosteric enzyme
behavior are called the concerted model and the
sequential model.
• In the concerted model, the enzyme is thought of as
being in a taut form, T, or a relaxed form, R. All
subunits are found in one or the other, and there is
an equilibrium between the T and R forms.
• Substrate binds more easily to the R form than to the
T form, inhibitors stabilize the T form, and activators
stabilize the R form
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Summary
Control of Enzyme Activity via Phosphorylation
• In the sequential model, subunits of the enzyme can
change sequentially from the T form to the R form
and back again.
• Binding of one molecule of substrate to one subunit
stimulates the transition of the subunit to the R form,
which then stimulates another subunit to change to
the R form.
• Binding of inhibitor to one subunit induces a change
in the other subunits to a form with lower affinity for
the substrate. Binding of an activator to one subunit
induces a shift in the other subunits to a form that has
a high affinity for substrate.
• The side chain -OH groups
of Ser, Thr, and Tyr can
form phosphate esters
• Two main types of protein kinases operate in
intracellular signaling pathways:
• Does Phosphorylation always increase enzyme
activity?
• Some enzymes are activated or inactivated,
depending on the presence or absence of phosphate
groups.
• This kind of covalent modification can be combined
with allosteric interactions to allow for a high degree
of control over enzymatic pathways.
• the most common are serine/threonine kinases,
which phosphorylate proteins on serines or threonines
• Others are tyrosine kinases, which phosphrylate
proteins on tyrosines.
Glycogen Phosphorylase Activity and
Allosteric Control
• Phosphorylation by ATP
can convert an inactive
precursor into an active
enzyme
• Membrane transport is a
common example
Membrane Transport: Na+-K+ pump
Na+ out, K+ in
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Summary
• Many enzymes are controlled by phosphorylation.
• Enzymes called kinases use high-energy molecules,
such as ATP, to transfer a phosphate to a specific
residue in an enzyme.
• These amino acid residues are usually serine,
threonine, or tyrosine residues.
• In some cases, phosphorylation increases the activity
of an enzyme, while in other cases it decreases it.
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
• Why is it necessary or advantageous for the body
make zymogens?
Zymogens are often seen with digestive enzymes
that are produced in one tissue and used in another.
If the enzyme were active immediately upon
production, it would digest other cell proteins, where
it would cause great damage.
By having it produced as a zymogen, it can be safely
made and then transported to the digestive tissue,
such as the stomach or small intestine, where it can
then be activated.
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 1°structure that accompany the change from
chymotrypsinogen to -chymotrypsin result in changes in
2°- and 3°structure as well.
• -chymotrypsin is enzymatically active because of its 2°and 3°structure, just as chymotrypsinogen was inactive
because of its 2°- and 3°structure
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The Nature of 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?
Non-Allosteric Enzyme Behavior:
Chymotrypsin
• Point at which the rate of
reaction does not change,
enzyme is saturated,
maximum rate of reaction
is reached
• 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
Chymotrypsin
• 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
released…free enzyme
is regenerated
• Reaction with a model substrate
Chymotrypsin (Cont’d)
Chymotrypsin (Cont’d)
• Chymotrypsin is a serine protease
• H57 also critical for
activation of enzyme
• Serine is required for the activity of chymotrypsin
• Trypsin and thrombin are also serine proteases
• DIPF inactivates chymotrypsin by reacting with
serine-195, verifying that this residue is at the active
site
• Can be chemically
labeled by TPCK
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Chymotrypsin (Cont’d)
Chymotrypsin (Cont’d)
• Because Ser-195 and His-57 are required for activity,
they must be close to each other in the active site
• The active site of chymotrypsin shows proximity of
reactive a.a.
• 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
Mechanism of Action of Critical Amino Acids in
Chymotrypsin
• Serine oxygen is nucleophile
• Nucleophile (nucleus-seeking): donate electron pair
• Attacks carbonyl group of peptide bond
• Electrophile(electron-seeking): accept electron pair
• General Base: accept proton
• General Acid: Donate proton
•His 57 accepts a hydrogen from Ser 195, acting as a general
Base
• Ser 195 has extra electron pair and nucleophilic attack
the carbonyl carbon of the peptide group.
•The proton abstracted by the histidine has been donated to the
Leaving amino group, His 57 acts as an acid
Fig. 7-14a, p. 181
His 57 accepts proton (base),
Water: nucleophilic attack on the acyl carbon that came from the
Original peptide bond.
Fig. 7-14b, p. 181
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• Explain why the second phase of the chymotrypsin
mechanism is slower than the first phase.
The first phase is faster for several reasons. The serine at
position 195 is a strong nucleophile for the initial nucleophilic
attack. It then forms an acyl-enzyme intermediate. In the
second phase, water is the nucleophile, and it takes time for
water to diffuse to the right spot to perform its nucleophilic
attack. It is also not as strong a nucleophile as the serine.
Therefore, it takes longer for water to perform its nucleophilic
attack and break the acyl-enzyme intermediate than it takes
for serine to create it.
• Briefly describe the role of nucleophilic catalysis
in the mechanism of the chymotrypsin reaction.
•
In the first step of the reaction, the serine hydroxyl
is the nucleophile that attacks the substrate peptide
bond.
Catalytic Mechanisms
Catalytic Mechanisms (Cont’d)
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.
•
In the second step, water is the nucleophile that
attacks the acyl-enzyme intermediate.
• 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
• same type of chemistry can occur at active site of
enzyme: SN1, SN2
Catalytic Mechanisms (Cont’d)
Enzyme Specificity
• Zn2+ of
carboxypeptidase is
complexed with:
• The imidazole side
chains of His-69 and
His-196 and the
carboxylate side
chain of Glu-72
• Absolute specificity: catalyzes the reaction of one unique
substrate to a particular product
• Activates the
carbonyl group for
nucleophilic acyl
substitution
• 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)
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Asymmetric binding
Active Sites and Transition States
• Enzymes can be
stereospecific
(Specificity where
optical activity may pay
a role)
• 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.
• Binding sites on enzymes
must be asymmetric
• Transition state analog: a substance whose shape mimics that of a
transition state
• In 1969 Jenks proposed that
Coenzymes
NAD+/NADH
• Coenzyme: a nonprotein substance that takes part in an
enzymatic reaction and is regenerated for further reaction
• metal ions- can behave as coordination compounds. (Zn 2+,
Fe2+)
• organic compounds, many of which are vitamins or are
metabolically related to vitamins (Table 7.1).
• Nicotinamide adenine
dinucleotide (NAD+) is used
in many redox reactions in
biology.
• 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
• Contains:
1) nicotinamide ring
2) Adenine ring
3) 2 sugar-phosphate groups
B6 Vitamins
• The B6 vitamins are coenzymes involved in amino group
transfer from one molecule to another.
• Important in amino acid biosynthesis
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