Micro-computer analysis of enzyme-catalyzed reactions by the Michaelis-Menten equation.

Abstract

Although Bardsley et al.1) theoretically and experimentally found that, in a very wide range of substrate concentrations, all enzymes probably deviate from the Michaelis-Menten equation and need equations of higher degrees, most enzyme kinetics have still been regarded to be Michaelian without serious limitations, and their kinetic constants such as the Michaelis constant (Km), maximum velocity (Vmax), and inhibition constant (Ki) have been calculated largely from the initial velocity data by the Lineweaver-Burk plot. Accordingly the poor reliability and reproducibility of Km and Vmax have widely been recognized for many enzymes, but an effective remedy has not yet been devised. To improve the present state of enzyme kinetics, we propose a practical method in which, with a micro-computer, an assay data set of an enzyme is critically checked at each data point for reasonable application of the differential Michaelis-Menten equation and then subjected to the calculation of Km and Vmax estimates by 5 methods. In particular, the assay data set composed of substrate concentration ([S0]) and initial reaction velocity (v) are corrected for substrate consumption, preferably by the method of Lee and Wilson.2) The whole data set is then checked for applicability of the Michaelis-Menten equation by three linear plots [Lineweaver-Burk (L-B), Hanes-Woolf (H-W), and Scatchard (SCAT) (or Eadie-Hofstee) plots] so that an unacceptable data pair(s), if present, can reasonably be deleted by visual examination. The trimmed data set is finally used for calculation of Km and Vmax by the three linear plots, the Wilkinson (WILK) method, and the nonparametric (NONP) method (or direct linear plot).