Basic kinetic measurements k1 s a0 s p step 1 write

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Unformatted text preview: e high affinity inhibitors with good “drug-like properties”. Basic Kinetic Measurements k1 S [A]0 [S] P Step 1. Write the reaction with the k's (rate constants) indicating the process. Step 2. Write how the compounds change with time: [P] d[S]/dt = - k1[S] d[P]/dt = k1[S] So, d[P]/dt = - d[S]/dt Velocity of the reaction is v and, v = d[P]/dt = - d[S]/dt = k1[S] (Units of k1 = sec-1) Enzyme Kinetic Experiment: S P 1. Use constant amount of enzyme, [ET]. 2. Measure amount of product P formed as a function of time with several initial concentrations of substrate S. 3. Calculate initial slopes from the graph of [P] vs. time to get initial rates, d[P]/dt = vo. 4. Plot the initial velocities as function of [S] [S]4 [P] [S]3 [S]2 [S]1 Concentration of Product P with time as function of increasing substrate concentrations [S]1, [S]2, [S]3, [S]4 Enzymes often form an Enzyme:Substrate complex E+S k1 k2 ES E+P k-1 E is enzyme; S is substrate; P is product; ES is the “Michaelis complex” For many enzyme-catalyzed reactions: ● Acceleration is very fast (µsec to msec) while the time scale is in minutes. ● d[ES]/dt = 0 means a “steady state condition” applies to most of the reaction progress. Total enzyme concentration [E]T is: [E]T = [E] + [ES] Vvp f12.02 Towards the Michaelis-Menten Equation E+S k2 k1 ES E+P (VVP eqn. 12-12) k-1 Where: k-1 k1 ES ES k2 Indicates the breakdown of ES. Indicates the buildup of ES. Assuming “steady state conditions” d[ES]/dt = 0 (VVP Eqn. 12-16) and defining the Michaelis constant KM: KM = (k-1+k2)/k1 (VVP Eqn. 12-20) one obtains for the initial velocity vo: vo = k2[ES] = k2 [ET][S]/(KM + [S]) (VVP eqn. 12-23) The Michaelis-Menten Equation The maximal velocity of a reaction, Vmax, occurs at high [S] when the enzyme is saturated, that is, when it is entirely in the [ES] form: Vmax = k2[ET] (VVP eqn 12-24). Subsituting this into : vo = k2[ET][S]/(KM + [S]) (VVP eqn. 12-23) ( i.e. the last eqn. at the previous slide) gives: vo = Vmax[S]/(KM + [S]) (VVP eqn. 12-25) this is the Michaelis-Me...
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