Lehninger Principles of Biochemistry 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 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, 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).

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

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:

I binds to E and ES with the same affinity (Ki = Ki) 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 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 Hydrophobic pocket Active site residues

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   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 diphosphate-ribosyl 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.