Basic kinetic measurements k1 s a0 s p step 1 write

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

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...
View Full Document

This note was uploaded on 10/20/2013 for the course BIOCHEM 405 taught by Professor Hol during the Fall '08 term at University of Washington.

Ask a homework question - tutors are online