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David L. Nelson and Michael M. Cox
Lehninger Principles of
Biochemistry
Fourth Edition
Chapter 6:
Enzymes (Part II)
Copyright © 2004 by W. H. Freeman & Company
[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-competitive
c) Mixed
(1) Irreversible Inhibition: inhibitor binds tightly, often
covalently, to the enzyme, permanently inactivating it.
DIPF = DIFP =
diisopropylfluorophosphate
(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.
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.
In competitive inhibition, inhibition can be overcome by
high [S].
Vmax does not change, but Km increases (Km,app = Km).
COO
CH2
OOC
H
succinate dehydrogenase
CH2
H
COO
COO
Fumarate
Succinate
COO
CH2
COO
Malonate
succinate dehydrogenase
No reaction
An uncompetitive inhibitor binds at a site other than
the active site and, binds only to the ES complex.
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 cannot be overcome by high [S].
Vmax,app – decrease
(by a factor of -1)
Km,app – decrease
(by a factor of -1)
Rare in single-substrate reaction.
More common in multisubstrate reaction
Ex) Compulsory ordered Bi-Bi reaction.
B
─BX
E + AX ⇄ EAX ⇄ EAXB ⇄ EABX ⇄ EA ⇄ E + A
EAXBI  No reaction
Compound, BI is an uncompetitive inhibitor of AX.
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:
v0 =
Vmax[S]
––––––––––
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 cannot be overcome by high [S].
Vmax,app – decrease (by a factor of (1 + [I]/Ki))
Km,app – unchanged
Ex) Compulsory ordered Bi-Bi reaction.
B
─BX
E + AX ⇄ EAX ⇄ EAXB ⇄ EABX ⇄ EA ⇄ E + A
B
EAXI ⇄ EAXIB
Compound, AXI is a noncompetitive inhibitor of B.
[7] Enzyme Mechanism - Chymotryipsin
Active
site
residues
Hydrophobic
pocket
Lehninger
p.216
Hexokinase and Induced Fit
[7] Enzyme regulation
The rates of enzyme-catalyzed reactions are altered by
activators and inhibitors (a.k.a. effector molecules).
(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, an
allosteric 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).
 In most cases, the first enzyme of the multireaction
pathway (catabolism, anabolism) is a regulatory enzyme to
avoid unneeded accumulation of the intermediates.
(2) Feedback inhibition: An enzyme,
early in the metabolic pathway, is
inhibited by an end-product. Often
takes place at the committed step
of the pathway, the step which commits
a metabolite to a pathway.
(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.
Catalytic domains
Catalytic domains
Catalytic domains
Regulatory domains
(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.
(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 enzyme’s
conformation and enhances the binding of
subsequent substrates.
⇒ Sigmoidal kinetics.
⇒ sensitive to a small change in [S].
(b) Heterotropic allosteric enzymes
(substrate = effector)
(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:
① Phosphate groups: cause a change in the 3D
structure
enhancing or inhibiting enzyme activity.
Enzymes are phosphorylated by a protein kinase or
dephosphorylated by a phosphatase.
Glycogen phosphorylase
(Glucose)n + Pi  (glucose)n-1 + glucose 1-Ⓟ
Glycogen
Shortened glycogen
② Adenylation: the transfer of adenylate from ATP
③ ADP-ribosylation: the transfer of an adenosine diphosphateribosyl moiety from NAD+
④ Uridylation
⑤ Methylation
(6) Proteolytic activation:
Some enzymes are synthesized as larger inactive precursor
forms called proenzymes or zymogens.
Activation involves the irreversible hydrolysis of one or more
peptide bonds, resulting in an active form.