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 CH2 COO Succinate COO CH2 COO Malonate succinate dehydrogenase succinate dehydrogenase

OOC

COO

Fumarate

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 EAX EAXB EABX EA E + A EAXBI 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 EAX EAXB EABX EA E + A

B EAXI EAXIB 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 enzymes 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: 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

 Adenylation: the transfer of adenylate from ATP ADP-ribosylation: the transfer of an adenosine diphosphateribosyl moiety from NAD+ Uridylation 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.