New standards for collecting and fitting steady state kinetic data

Beilstein J Org Chem. 2019 Jan 2:15:16-29. doi: 10.3762/bjoc.15.2. eCollection 2019.

Abstract

The Michaelis-Menten equation is usually expressed in terms of k cat and K m values: v = k cat[S]/(K m + [S]). However, it is impossible to interpret K m in the absence of additional information, while the ratio of k cat/K m provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, k cat/K m provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis-Menten equation in terms of k cat and k cat/K m values: v = k SP[S]/(1 + k SP[S]/k cat), where the specificity constant, k SP = k cat/K m. In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of k cat/K m can be derived directly using the modified form of the Michaelis-Menten equation rather than calculated from the ratio of k cat and K m values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive k cat and k cat/K m is illustrated by considering the role of conformational changes in enzyme specificity where k cat and k cat/K m can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret K m, which is best understood as the ratio of k cat divided by k cat/K m.

Keywords: Michaelis constant; computer simulation; data fitting; enzyme catalysis; induced-fit; specificity constant.

Publication types

  • Review