Kinetic Applications Using High Substrate and Competitive Inhibitor Concentrations to Determine Ki or Km

Abstract

Conventional procedures for determining Km or Ki values generally employ subsaturating concentrations of substrate and competitive inhibitor; however, this is impractical in some cases if a significant percentage would be enzyme-bound or metabolized. Applications employing high and competing concentrations of substrates and inhibitors are described. Ki or Km values can be calculated from the equationsKi = [I]/((V0)/V - 1)(1 + [S]/Km))Km = [S](V0/V - 1)/(1 - V0/V + [I]/Ki),where V0 and V are the rates in the absence and presence of inhibitor. This is illustrated by measurements of inhibition of human urinary kallikrein by synthetic peptides. © 1994 Academic Press. All rights reserved.