Crompton I E, Waley S G
Biochem J. 1986 Oct 1;239(1):221-4. doi: 10.1042/bj2390221.
A convenient and accurate procedure for determining the kinetic parameter Vmax./Km is described. This avoids the error in the usual method of taking the observed first-order rate constant of an enzymic reaction at low substrate concentration as Vmax./Km. A series of reactions is used in which the initial concentration of substrate is below Km (e.g. from 5% to 50% of Km). Measurements are taken over the same extent of reaction (e.g. 70%) for each member of the series, and treated as if the kinetics were truly first-order. The reciprocal of the observed first-order rate constant is then plotted against the initial concentration of substrate: the reciprocal of the ordinate intercept is Vmax./Km. The procedure, as well as being applicable to simple reactions, is shown to be valid when there is competitive inhibition by the product, or when the reaction is reversible, or when there is competitive or mixed inhibition. The hydrolysis of cephalosporin C by a beta-lactamase from Pseudomonas aeruginosa is used to illustrate the method.
本文描述了一种便捷且准确的测定动力学参数Vmax./Km的方法。该方法避免了常规方法中的误差，即在低底物浓度下将酶促反应的观测一级速率常数当作Vmax./Km。使用了一系列反应，其中底物的初始浓度低于Km（例如为Km的5%至50%）。对该系列中的每个反应成员，在相同的反应程度（例如70%）范围内进行测量，并当作动力学为真正的一级反应来处理。然后将观测一级速率常数的倒数对底物的初始浓度作图：纵坐标截距的倒数即为Vmax./Km。该方法不仅适用于简单反应，还表明在存在产物竞争性抑制、反应可逆、存在竞争性或混合型抑制的情况下也是有效的。以铜绿假单胞菌的β-内酰胺酶催化头孢菌素C的水解反应为例来说明该方法。
