Evaluation of a minimal experimental design for determination of enzyme kinetic parameters and inhibition mechanism

The advent of combinatorial chemistry has led to a deluge of new chemical e ntities whose metabolic pathways need to be determined. A significant issue involves determination of the ability of new agents to inhibit the metabol ism of existing drugs as well as its own susceptibility for altered metabol ism. There is need to estimate the enzyme inhibition parameters and mechani sm or mechanisms of inhibition with minimal experimental effort. We examine d a minimal experimental design for obtaining reliable estimates of K-i (an d V-max and K-m). Simulations have been applied to a variety of experimenta l scenarios. The least experimentally demanding case involved three substra te concentrations, [S], for the control and one substrate-inhibitor pair, [ S]-[I]. The control and inhibitor data (with 20% coefficient of variance ra ndom error) were simultaneously fit to the full nonlinear competitive inhib ition equation [simultaneous nonlinear regression (SNLR)]. Excellent estima tes of the correct kinetic parameters were obtained. This approach is clear ly limited by the a prior assumption of mechanism. Further simulations dete rmined whether SNLR would permit assessment of the inhibition mechanism (co mpetitive or noncompetitive). The minimal design examined three [S] (contro l) and three [S]-[I] pairs. This design was successful in identifying the c orrect model for 98 of 100 data sets (20% coefficient of variance random er ror). SNLR analysis of metabolite formation rate versus [S] permits a drama tic reduction in experimental effort while providing reliable estimates of K-i, K-m, and V-max along with an estimation of the mechanism of inhibition . The accuracy of the parameter estimates will be affected by the experimen tal variability of the system under investigation.