One Enzyme One Substrate一种酶一底物.PPT

Enzyme Kinetics Mary V. Relling, Pharm.D. Carl Panetta, Ph.D. Learning Objectives: Understand the Michaelis-Menten equation and the most common methods of plotting reaction rates vs substrate or inhibitor concentrations. Be familiar with the basic assumptions required to apply that equation to simple experimental drug metabolism models. Learning Objectives Continued: List at least 3 factors that could affect estimates of Km, including the basic effects of competitive inhibitors. Understand the difference between Ki, Km, and IC50. Be able to choose reasonable starting concentrations for substrates or inhibitors to set up an experiment to determine Ki, Km, and Vmax, given basic limiting conditions. One Enzyme One Substrate Simplify System Quasi-Steady-State Assumption Assume [S]>>[E]---enzyme is working at capacity therefore occupancy rate is ~ constant. Thus: Michaelis-Menten Equation(after a bit of algebra) Experimental implications of Quasi-Steady-State Assumption Only initial velocities should be measured, i.e. substrate concentration is high relative to product concentration (should be < 10% disappearance of substrate) Substrate: Enzyme ratio should be high (> 100:1). Comments on experiments at initial velocity Ensure V is linear with time (constant substrate, enzyme, cofactors). Ensure V is linear with protein (constant substrate, time, cofactors) Ensure [S] is informative (constant enzyme, time, cofactors). Vmax include S at KM/2 to 5*KM (at least 5 data pts) Consider possibility of two ezs: high KM and low KM See later comments on sampling strategies Comments on experiments at initial velocity To estimate kinetic parameters Invariant and linear conditions for Time Temperature co-factors amount of enzyme Vary substrate [S]---(e.g. mM)--encompass Km(s) Measure V---(e.g. nmol/mg protein/min) Limitations: Very low [S] results in undetectable product Very high [S] may be insoluble--or cause product inhibition Methods of estimating parameters Use any program th