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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

pocket

[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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