Waley S G
University of Oxford, New Chemistry Laboratory, Oxford Centre for Molecular Sciences, U.K.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):195-200. doi: 10.1042/bj2940195.
Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.
解离常数在微摩尔至纳摩尔范围内的抑制剂很重要，但在动力学上难以表征，尤其是当测定中的底物浓度低于米氏常数（Km）时。当在测定过程中抑制作用增强（慢结合抑制）时，底物浓度可能会显著降低。本文描述了考虑底物消耗的方法，用于分析初始底物浓度低于Km的实验。拟合进程曲线可得出两步机制中第二步（慢）步骤的速率常数。当将反应视为一级过程时，可通过速率测量确定总解离常数的近似值。当抑制剂和酶的浓度相当时，则需要数值方法。本文还描述了适用于在微型计算机上实现的求解微分方程和拟合进程曲线的程序。
