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Lehninger Principles ofBiochemistry
Fourth Edition
Chapter 6:
Enzymes (Part II)
Copyright 2004 by W. H. Freeman & Company
David L. Nelson and Michael M. Cox
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[6] Enzyme Inhibition
Inhibitor: Any molecule that acts directly on an enzyme to lower its
catalytic rate. These can be cellular metabolites, or foreign
substances such as drugs or toxins that have either a therapeutic or
toxic (can be lethal) effect.
There are two major types of inhibition:
(1) Irreversible inhibition
(2) Reversible inhibition
a) Competitive
b) Un-competitivec) Mixed
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(1) Irreversible Inhibition: inhibitor binds tightly, often
covalently, to the enzyme, permanently inactivating it.
DIPF = DIFP =
diisopropylfluorophosphate
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(2) Reversible Inhibition
(a) Competitive inhibition:Inhibitor has close structural similarities to the normal
substrate and therefore competes with the substrate for the
active site.
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In the presence of a competitive inhibitor, I,
Vmax [S]
v0 =
Km(1 + [I]/Ki) + [S][E][I]
where Ki (inhibition constant) =[EI]
Then, Vmax [S]
v0 =Km+ [S]
where = (1 + [I]/Ki)
The type of inhibition can be
determined using the double reciprocal plot.
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In competitive inhibition, inhibition can be overcome by
high [S].
Vmax does not change, but Km increases (Km,app = Km).
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COO
CH2
CH2
COO
succinate dehydrogenaseOOC H
H COO
SuccinateFumarate
COO
CH2succinate dehydrogenase
Malonate
COONo reaction
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An uncompetitive inhibitor binds at a site other than
the active site and, binds only to the ES complex.
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v0 = Vmax [S]
Km + [S] where = (1 + [I]/Ki)and Ki
= [ES][I]/[ESI].
Since I does not share the binding site with S,
uncompetitive inhibition cannotbe overcome by high [S].
Vmax,app decrease(by a factor of-1)
Km,app decrease
(by a factor of-1)
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Ex) Compulsory ordered Bi-Bi reaction.
B BXE + AX EAX EAXB EABX EA E + A
EAXBI No reaction
Compound, BI is an uncompetitive inhibitor of AX.
Rare in single-substrate reaction.
More common in multisubstrate reaction
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Inhibitor binds at a site other than the active site (E or ES)and causes changes in the overall 3-D shape of the enzyme
that leads to a decrease in activity:
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Vmax[S]
v0 =
Km + [S]
where = (1 + [I]/Ki) and
= (1 + [I]/Ki)
Ki = [E][I]/[EI],
Ki= [ES][I]/[ESI].
When, = , that is,
I binds to E and ES with the same affinity (Ki = Ki)
Noncompetitive inhibition.
Mixed inhibition cannotbe overcome by high [S].
Vmax,app decrease (by a factor of (1 + [I]/Ki))
Km,app unchanged
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Ex) Compulsory ordered Bi-Bi reaction.
B BX
E + AX EAX EAXB EABX EA E + A
B
EAXI EAXIB
Compound, AXI is a noncompetitive inhibitor of B.
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Active
site
residues
Hydrophobic
[7] Enzyme Mechanism - Chymotryipsin
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Lehninger
p.216
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Hexokinase and Induced Fit
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[7] Enzyme regulation
The rates of enzyme-catalyzed reactions are altered by
activators and inhibitors (a.k.a. effectormolecules).
(1) Allosteric enzymes: have more than one site, where
effector binding at one site induces a conformational
change in the enzyme, altering its affinity for a substrate.
An allosteric activator increases enzyme rate of activity, anallosteric inhibitor decreases its activity.
Regulation mechanism:
Reversible, noncovalent binding of allosteric effectors.
Covalent modification (phosphorylation, adenylation, etc.).
Binding by separate regulatory proteins.
Proteolytic activation (irreversible).
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In most cases, the first enzyme of the multireaction
pathway (catabolism, anabolism) is a regulatory enzyme to
avoid unneeded accumulation of the intermediates.
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(2) Feedback inhibition: An enzyme,
early in the metabolic pathway, is
inhibited by an end-product. Oftentakes place at the committed step
of the pathway, the step which commits
a metabolite to a pathway.
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(3) Regulatory enzymes are generally more complex than other
enzymes,
i.e.Aspartate transcarbamoylase first step in CTP synthesis,
converts Asp to N-carbamoyl Asp
CO2 + Gln + ATP H2N-(C=O)-OPO32-
(carbamoyl phosphate)
Asp transcarbamoylase catalyzes the following reaction:Carbamoyl phosphate + Asp N-carbamoylAspartate
CTP (building block of DNA)
CTP, the end product of the reaction, decreases the rate of
enzyme activity allosteric inhibitor.ATP increases the rate of enzyme activity allosteric activator.
Many effectors work in concert to regulate the pathway.
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Catalytic domains
Catalytic domains
Catalytic domains
Regulatory domains
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(4) Kinetic properties of regulatory enzymes
The relationship between enzyme velocity and substrate
concentration is often a sigmoidal saturation curve for an
allosteric enzyme rather than hyperbolic (Michaelis), and we
no longer refer to substrate concentration at half maximal
velocity as Km, we use [S]0.5 or K0.5.
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(a) Homotropic allosteric enzymes (substrate = effector):
- Multisubunit enzymes.
- The same binding site on each subunit
functions as both active site and regulatory site.- Substrate acts as an activator as well. (O2 and Hb).
- Binding of one substrate alters the enzymes
conformation and enhances the binding of
subsequent substrates. Sigmoidal kinetics. sensitive to a small change in [S].
(b) Heterotropic allosteric enzymes
(substrate = effector)
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(5)Reversible Covalent Modification: is the making and
breaking of a covalent bond between a non-protein group
and an enzyme that affects its activity.
Examples of some transfer groups:
Phosphate groups: cause a change in the 3Dstructure
enhancing or inhibiting enzyme activity.Enzymes are phosphorylated by a protein kinase or
dephosphorylated by a phosphatase.
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Glycogen phosphorylase
(Glucose)n + Pi (glucose)n-1 + glucose 1-
Glycogen Shortened glycogen
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Adenylation: the transfer of adenylate from ATP
ADP-ribosylation: the transfer of an adenosine diphosphate-
ribosyl moiety from NAD+
Uridylation Methylation
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(6) Proteolytic activation:
Some enzymes are synthesized as larger inactive precursor
forms called proenzymes orzymogens.
Activation involves the irreversible hydrolysis of one or morepeptide bonds, resulting in an active form.
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