Enzyme Kinetics Substrate affects rate and it changes

Enzyme Kinetics [Substrate] affects rate and it changes during reaction Can measure just initial rate, Vo, when [S]>>[E] k 2 k 1 E + S ES k-1 E + P k-2 Slow step Rate-limiting maximum velocity Velocity (V) = k [S]

Enzyme Kinetics Michaelis-Menten equation k 2 k 1 E + S ES E + P k-2 k-1 Derive: Assume that [P] low at start and that k-2 is very small V 0 = k 2[ES] hard to measure, so [Et] used [Et] = [E] + [ES], [E] = [Et] - [ES] small because [S] so large, [Et] = [E] Formation of ES = k 1([Et] - [ES])[S] Breakdown of ES = k-1[ES] + k 2[ES] Assume steady state [ES] ~ constant, so k 1([Et] - [ES])[S] = k-1[ES] + k 2[ES] Rearrange: k 1[Et][S] = (k 1[S] + k-1 + k 2) [ES] = k 1[Et][S] (k 1[S] + k-1 + k 2)

Enzyme Kinetics Michaelis-Menten [ES] = V 0 = k 1[Et][S] (k 1[S] + k-1 + k 2) [Et][S] + (k-1 + k 2) / k 1 [Et][S] + Km k 2 [Et][S] + Km Vmax[S] + Km Km = (k-1 + k 2) / k 1 Km = Michaelis constant Remember V 0 = k 2[ES] Vmax occurs when [ES] = [Et] Vmax = k 2[Et] Michaelis-Menten equation!! Rate equation for 1 substrate, enzyme-catalyzed reaction Km has units of concentration

Enzyme Kinetics Michaelis-Menten Vmax[S] V 0 = [S] + Km When V 0 = 1/2 Vmax / 2 = Vmax[S] 1/2= [S] + Km Km = [S] Low [S], Km >> [S] V 0 = High [S], [S] >> Km V 0 = Vmax[S] Km Vmax

Enzyme Kinetics Transform Michaelis-Menten Equation V 0 = Vmax[S] + Km Km 1 1 + V 0 = Vmax[S] Vmax Double reciprocal or Lineweaver-Burk plot 1/V vs. 1/[S] Straight line --> slope, y-intercept, x-intercept More accurate determination of Vmax

Enzyme Kinetics Km and kcat Km = k 2 + k-1 k 1 Km measurement --> affinity of enzyme for its substrate kcat = Vmax/[E]T kcat is catalytic constant or turnover number (first order rate constant, s-1) CATALYTIC EFFICIENCY

Enzymes can be Inhibited Competitive inhibitor competes with substrate for active site Noncompetitive inhibitor binds elsewhere, influencing binding at active site

Enzymes can be Inhibited Competitive Inhibition Active site of enzyme Substrate Inhibitor Substrate and Inhibitor can bind to active site Inhibitor prevents binding of substrate

Enzymes can be Inhibited Noncompetitive Inhibition Active site of enzyme Substrate Inhibitor site Substrate can bind to active site product forms Inhibitor binding distorts active site Inhibitor and substrate can bind simultaneously, rate slowed

Enzymes can be Inhibited Competitive Inhibition Competitive inhibitor Apparent Km will increase No effect on Vmax Increasing concentration of inhibitor -1/ Km

Enzymes can be Inhibited Competitive Inhibition Ingestion of methanol (gas-line antifreeze) In liver, alcohol dehydrogenase converts methanol to formaldehyde (BAD) Ethanol competes effectively with methanol for binding to alcohol dehydrogenase Therapy for methanol poisoning is IV with ethanol, formaldehyde not formed as readily, little tissue damage, kidneys excrete methanol

Enzymes can be Inhibited Noncompetitive Inhibition +Noncompetitive inhibitor 1/V No inhibitor -1/Km 0 1/[S] Noncompetitive inhibitor Apparent Km not affected Lowering of Vmax

Enzymes can be Inhibited Noncompetitive Inhibition Also called allosteric inhibition Example of noncompetitive inhibitor = aspirin Aspirin inhibits a cyclo-oxygenase so that prostaglandins may not be synthesized, thereby reducing pain, fever, inflammation, blood clotting, etc. Aspirin does not bind to the active site of cyclo-oxygenase but to a separate/allosteric site

Enzymes can be Inhibited Irreversible Inhibition - Inhibitor binds covalently to or destroys essential functional group on enzyme Suicide inactivators undergoes first few steps of rxn and then converts to a reactive compound that combines irreversiby with enzyme (high specificity) INHIBITS ornithine decarboxylase (cure for African sleeping sickness)

Enzymes can be Inhibited Enzymes inhibitors act only at the active site of an enzyme: 1. 2. False True

Enzymes can be Inhibited Enzymes inhibitors designed to “look” like the substrate will act on: 1. 2. 3. 4. An allosteric site on the enzyme The enzyme active site The substrate None of the above

Enzymes can be Inhibited Enzymes inhibitors bind to the enzyme through 1. 2. 3. 4. Covalent bonds Noncovalent bonds Both of the above Neither of the above