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.
Skip Nav Destination
Research Article| October 01 1986
The determination of specificity constants in enzyme-catalysed reactions
Biochem J (1986) 239 (1): 221–224.
- Views Icon Views
- Share Icon Share
I E Crompton, S G Waley; The determination of specificity constants in enzyme-catalysed reactions. Biochem J 1 October 1986; 239 (1): 221–224. doi: https://doi.org/10.1042/bj2390221
Download citation file:
Don't already have an account? Register
Get Access To This Article
Buy This Article