Nonlinear determination of Michaelis-Menten kinetics with model evaluation through estimation of uncertainties.

A structured analytic approach was developed to evaluate enzyme kinetic parameters using non-linear least-squares fitting. The approach was implemented in a software package called KinSim and designed to run on Windows 95, 98, and NT platforms. The software and the theoretical approach were tested using kinetic data obtained using citrate synthase with acetyl CoA as a substrate and octanoyl CoA as an inhibitor. Using the software, the data were evaluated for statistical certainty of the presence of competitive and non-competitive inhibition of the enzyme. Given the determination of the presence of both components of inhibition in the experimental setup, the software was used to determine the inhibitory kinetics from the experimental data, including non-normal distributions of uncertainty. Finally, the software was used to evaluate the sensitivities of the experiment design at each concentration of substrate used. The theoretical approach as implemented in the user-friendly software allows investigators to use a structured procedure for evaluating and planning enzyme kinetic and related metabolic studies.