Enzymes - Binding and Catalysis

ENZYMES: Binding & Catalysis A. B. C. D. E. Binding Catalysis Nomenclature Catalysis 1. 2. 3. a. b. c. Transition State Theory Catalytic strategies (What) Mechanistic strategies (How) 7. 1. 2. 3. 4. b. 5. 6. b. c. Review Enzyme Kinetics Rate vs. [S] for enzyme catalyzed reaction; initial rate (!0) ES complex a. Meaning of rate expression (M-M Irreversible: protein modification Reversible i. Competitive; like substrate; Km affected by (1 + [I]/KI) = a ii. Uncompetitive; binds only ES; both Km and Vmax affected in opposite ways iii. Noncompetitive; binds both E & ES (mixed, non-equal binding); Vmax affected iv. Mixed inhibition if I binds E differently than it binds ES Meaning of rate curve: hyperbolic curve Assumptions M-M equation derivation Lineweaver-Burk; double reciprocal; 1/v0 vs. 1/[S] Eadie-Hofstee; v0 vs. v0/[S] Hanes-Woolf; [S]/v0 vs. 1/[S] Inhibition a. b. Binding reaction Catalytic reaction Rate expression; Michaelis-Menten Kinetics (M-M) a. b. 8. Reaction i. ii. Collection and manipulation of data a. Quantifying the Catalytic Power: Kinetics [S] = Km [S] >> Km [S] << Km v. vi. Using Steady-state kinetics: Active Site Energetics of Catalysis eqn) Enzyme Kinetics Determination of Kinetic Parameters Slopes = v0 (rates) A nonlinear Michaelis-Menten plot could be used to calculate parameters Km and Vmax. Lineweaver-Burk derived a linear form of the M-M equation by taking the reciprocal of both sides. This is called the linearized double-reciprocal plot. Its good for analysis of enzyme kinetic data to get these kinetic parameters. Enzyme Kinetics Lineweaver-Burk Plot: Linearized, Double-Reciprocal The Michaelis-Menten equation can be manipulated into one that yields a straight-line plot. !0 = Vmax [S] Km + [S] y = bx + a y= 1 !0 x= 1 [S] 1 K + [S] !0 = m Vmax [S] 1 Km [S] + Vmax [S] Vmax [S] 1 1 Km + Vmax [S] Vmax !0 = !0 = This double-reciprocal equation is called the Lineweaver-Burk equation. Enzyme Kinetics Other Linearized Derivations of the M-M Equation Hanes-Woolf Eadie-Hofstee !0 = Vmax – Km !0 [S] [S] vs. [S] !0 !0 vs. !0 [S] [S]/v0 Vmax v0 –Km Vmax/Km [S] v0/[S] Enzyme Kinetics Linearized Derivations of the M-M Equation Lineweaver-Burk Hanes-Woolf Eadie-Hofstee !0 = Vmax – Km !0 [S] [S] vs. [S] !0 !0 vs. !0 [S] [S]/v0 Vmax v0 –Km Vmax/Km [S] !0 = v0/[S] Vmax [S] Km + [S] Enzyme Kinetics ENZYME INHIBITION Enzyme Kinetics What is Enzyme Inhibition? This is the action of a small molecule that results in loss of enzyme activity This is not regulation by the action of another enzyme or protein This is not loss of enzyme activity due to denaturation/unfolding of the enzyme. Two major kinds of inhibition 1) Irreversible 2) Reversible Enzyme Kinetics Reversible Enzyme Inhibition: There are THREE-to-FOUR types of reversible inhibition: 1) 2) 3) 4) Competitive Un-Competitive Non-Competitive Mixed These are closely related Enzyme Kinetics Reversible Enzyme Inhibition: Km (Binding) Vmax (Catalysis) k1 k2 E+S⇌ ES ⇀ E + P k -1 No Inhibition Enzyme Kinetics Reversible Enzyme Inhibition: Km (Binding) Vmax (Catalysis) k1 k2 E+S⇌ ES ⇀ E + P k + I ⇌ E•I Competitive -1 Enzyme Kinetics Reversible Enzyme Inhibition: Km (Binding) Vmax (Catalysis) k1 k2 E+S⇌ ES ⇀ E + P k -1 + I ⇌ ES•I Un-Competitive Enzyme Kinetics Reversible Enzyme Inhibition: Km (Binding) Vmax (Catalysis) k1 k2 E+S⇌ ES ⇀ E + P k + I -1 + I ⇌ ⇌ E•I ES•I Non-Competitive Enzyme Kinetics Reversible Enzyme Inhibition: Km (Binding) Vmax (Catalysis) k1 k2 E+S⇌ ES ⇀ E + P k -1 + I E•I ES•I ⇌ ⥄ ⇌ ⥄ + I Mixed Enzyme Kinetics Reversible Enzyme Inhibition: There are THREE-to-FOUR types of reversible inhibition: 1) 2) 3) 4) Competitive Un-Competitive Non-Competitive Mixed These are closely related Non-competitive Inhibition • Binds BOTH free enzyme (E) and enzyme bound to substrate (ES) ― binds to a entirely different site from the active site (regulatory/inhibitory site) ― Essentially just titrates out the [enzyme] ― inhibits both substrate binding and catalysis equally (rare) Non-competitive inhibition How does this inhibition affect the rate expression? Affects Catalysis; Binding is affected but to the same degree in opposite ways, so it cancels. Which kinetic constant will be affected? Apparent Km stays the same as its pulled and pushed the same. Apparent Vmax gets smaller due to loss of [E]T: ([E]+[ES]+[EI]+[ESI]). Non-competitive Inhibition Non-competitive inhibition !0 = !0 = [E][I] [E]K = I [EI] [ES][I] [ES]K = I [ESI] [E]K I = [ES]KI !0 = Vmax [S] Km + [S] Vmax [S] (1+ [ I ] ) KI Km + [S] Vmax [S] (Km + [S]) •(1+ [ KI I] ) SUMMARY: Reversible Inhibition Competitive = got worse App Km = got better App Vmax Km (Binding) Vmax (Catalysis) E+S + k1 k-1 ES I + I E•I ES•I k2 E+P Km (1+ [KI ] ) Vmax I Un-Competitive Km Vmax ( 1+ [K’I ] ) ( 1+ [K’I ] ) Km Vmax ( 1+ [K’I ] ) I I Non-Competitive [I] 1+ KI ( )=a (1+ [K’I ]I ) = a’ I Mixed Km (1+ [KI ] ) I ( 1+ [K’I ] ) I Vmax ( 1+ [K’I ] ) I Enzymes SUMMARY SO FAR: ACTIVE SITE We have described enzymes in general terms such as: catalytic cycle binding, even stereo-specific binding catalysis, turnover number & proficiency nomenclature transition state theory catalytic strategies (what to do) mechanistic strategies (how to do) enyzme kinetics and inhibition ALL of this happens at the ACTIVE SITE Now, we want to ask what all happens here & how do we determine what happens? Enzymes How do your determine what is going on at the active site? We will discuss FOUR methods for study of the active site 1. 2. 3. 4. Enzyme kinetics pH studies Protein modification Structural studies