The main contents of this chapter include all of the following: Lineweaver Burk plots, mutlisubstrate reactions, inhibitor kinetics, Irreversible inhibitors – know the differences between a reversible and irreversible inhibitor. | Chapter 5 (part 3) Enzyme Kinetics (cont.) Michaelis-Menton Vmax Km Kcat Kcat/Km E + S ES E + P k1 k-1 kcat Vo = Vmax [S] Km + [S] Vmax Km kcat kcat/Km How do you get values for Vmax, Km and kcat? Can determine Km and Vmax experimentally Km can be determined without an absolutely pure enzyme Kcat values can be determined if Vmax is known and the absolute concentration of enzyme is known (Vmax = kcat[Etotal] Lineweaver-Burke Plots (double reciprocal plots) Plot 1/[S] vs 1/Vo L-B equation for straight line X-intercept = -1/Km Y-intercept = 1/Vmax Easier to extrapolate values w/ straight line vs hyperbolic curve V max Km Km ~ mM Vmax ~ -1/Km = Km = mM 1/Vmax = Vmax = Enzyme Inhibition Inhibitor – substance that binds to an enzyme and interferes with its activity Can prevent formation of ES complex or prevent ES breakdown to E + P. Irreversible and Reversible Inhibitors Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly) Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme) Why important? Reversible Inhibitors E + S ES -> E + P E + I EI Ki = [E][I]/[EI] Competitive Uncompetitive Non-competitive Types of Reversible Enzyme Inhibitors Competitive Inhibitor (CI) CI binds free enzyme Competes with substrate for enzyme binding. Raises Km without effecting Vmax Can relieve inhibition with more S Competitive Inhibitors look like substrate PABA Sulfanilamide PABA precursor to folic acid in bacteria O2C-CH2-CH2-CO2 -------> O2C-CH=CH-CO2 succinate fumarate Succinate dehydrogenase O2C-CH2-CO2 Malonate Uncompetitive Inhibitor (UI) UI binds ES complex Prevents ES from proceeding to E + P or back to E + S. Lowers Km & Vmax, but ratio of Km/Vmax remains the same Occurs with multisubstrate enzymes Non-competitive Inhibitor (NI) NI can bind free E or ES complex Lowers Vmax, but Km remains the same NI’s don’t bind to S binding site therefore don’t effect Km Alters conformation of enzyme to effect catalysis but not substrate binding Irreversible Inhibitors Diisopropyl fluorophosphate (nerve gas) parathion malathion Organophosphates Inhibit serine hydrolases Acetylcholinesterase inhibitors Viagra Kinetics of Multisubstrate Reactions E + A + B E + P + Q Sequential Reactions ordered random Ping-pong Reactions Cleland Notation Sequential Reactions E EA (EAB) (EPQ) EQ E A B P Q A B P Q A B E EA EB (EAB)(EPQ) P Q EQ EP E Ordered Random Ping-Pong Reactions E (EA)(FP) (F) (FB)(EQ) E A B P Q In Ping-Pong rxns first product released before second substrate binds When E binds A, E changes to F When F binds B, F changes back to E Lineweaver-Burke Plot of Multisubstrate Reactions Increasing [B] Increasing [B] Sequential Ping-Pong Vmax doesn’t change Km changes Both Vmax & Km change 1/Vo 1/[S] 1/Vo 1/[S]
