Substrate inhibition kinetics for cytochrome P450-catalyzed reactions

Most cytochrome P450 (P450 or CYP)-catalyzed reactions are adequately described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), which are usually determined by a saturation profile of velocity of product formation versus substrate concentration. In turn, these parameters may be used to predict pharmacokinetics. However, some P450 enzymes exhibit atypical or non-Michaelis-Menten kinetics, due largely to substrate inhibition at higher concentrations of substrate. Although the mechanism of substrate inhibition is unknown, ignoring it and truncating the data can lead to erroneous estimates of kinetic parameters. In the present study, 13 P450 marker substrates were examined with 10 recombinant P450 proteins, and 6 were found, to varying degrees, to exhibit substrate inhibition. To understand the nature of the inhibition, a kinetic model was proposed (assuming that two binding sites exist on the enzyme) and used to fit the experimental data. The derived data indicated that 1) the K(I) values (substrate inhibition) were approximately 1.2- to 10-fold greater than the respective K(S) values; 2) both K(S) and K(I) values may be affected by the interaction of the two bound substrates within the enzyme, exhibited by a factor alpha (alpha = 5.1-23.3); and 3) enzyme activity was inhibited markedly (39-97%) at excess concentrations of the substrates (beta = 0.03-0.61). These findings suggest that substrates have access to both the inhibitory site and catalytic site simultaneously (K(I) > K(S)). Furthermore, the two sites, in the presence of substrate, can interact with each other. Therefore, the degree of inhibition of the enzyme is dependent on the concentration of the substrate (usually >K(I)) that sufficiently occupies the inhibitory site.