Extracting Kinetic Isotope Effects From a Global Analysis of Reaction Progress Curves.

Abstract

Enzyme reaction progress curves, or time course datasets, are often rich in information, yet their analysis typically reduces their information content to a single parameter, the initial velocity. An alternative approach is described here, where the time course is described by a model constructed from rate equations. In combination with global nonlinear regression, intrinsic rate and/or equilibrium constants can be directly obtained by fitting these data. This method can be greatly enhanced when combined with the measurement of (usually deuterium) isotope effects, which selectively perturb individual step(s) within the reaction, allowing better separation of fitted parameters and more robust model testing. This chapter focuses on practical considerations when using analytical and/or numerically integrated rate equations to model enzyme reactions. The emphasis is on the underlying methodology, which is demonstrated with specific examples alongside recommendations of suitable software.