2  Enzyme InhibitionInhibitor: 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 inhibitiona) Competitiveb) Un-competitivec) Mixed
3 (1) Irreversible Inhibition: inhibitor binds tightly, often covalently, to the enzyme, permanently inactivating it.DIPF = DIFP = diisopropylfluorophosphate
4 (2) Reversible Inhibition Competitive inhibition:Inhibitor has close structural similarities to the normal substrate and therefore competes with the substrate for the active site.
5 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 bedetermined using the double reciprocal plot.
6 In competitive inhibition, inhibition can be overcome by high [S]. Vmax does not change, but Km increases (Km,app = Km).
8 An uncompetitive inhibitor binds at a site other than the active site and, binds only to the ES complex.
9 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
10 Rare in single-substrate reaction. More common in multisubstrate reactionEx) Compulsory ordered Bi-Bi reaction.B ─BXE + AX ⇄ EAX ⇄ EAXB ⇄ EABX ⇄ EA ⇄ E + AEAXBI No reactionCompound, BI is an uncompetitive inhibitor of AX.
11 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:
12 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
13 Ex) Compulsory ordered Bi-Bi reaction. B ─BXE + AX ⇄ EAX ⇄ EAXB ⇄ EABX ⇄ EA ⇄ E + ABEAXI ⇄ EAXIBCompound, AXI is a noncompetitive inhibitor of B.
29  Enzyme regulationThe 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).
30 In most cases, the first enzyme of the multireaction pathway (catabolism, anabolism) is a regulatory enzyme to avoid unneeded accumulation of the intermediates.
31 (2) Feedback inhibition: An enzyme, early in the metabolic pathway, isinhibited by an end-product. Oftentakes place at the committed stepof the pathway, the step which commitsa metabolite to a pathway.
32 (3) Regulatory enzymes are generally more complex than other enzymes, i.e. Aspartate transcarbamoylase – first step in CTP synthesis, converts Asp to N-carbamoyl AspCO2 + 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.
34 (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.
35 (a) Homotropic allosteric enzymes (substrate = effector): - Multisubunit enzymes.- The same binding site on each subunitfunctions as both active site and regulatory site.- Substrate acts as an activator as well. (O2 and Hb).- Binding of one substrate alters the enzyme’sconformation and enhances the binding ofsubsequent substrates.⇒ Sigmoidal kinetics.⇒ sensitive to a small change in [S].(b) Heterotropic allosteric enzymes(substrate = effector)
36 (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 structureenhancing or inhibiting enzyme activity.Enzymes are phosphorylated by a protein kinase ordephosphorylated by a phosphatase.
38 ② Adenylation: the transfer of adenylate from ATP ③ ADP-ribosylation: the transfer of an adenosine diphosphate-ribosyl moiety from NAD+④ Uridylation⑤ Methylation
39 (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.