Sigmoidal kinetic model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an example of the metabolism of diazepam and its derivatives

Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificit ies. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multi ple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) ch aracteristics that make the Michaelis constants difficult to estimate. In t he present study, diazepam, temazepam and nordiazepam were employed as subs trates of CYP3A4 to propose a kinetic model. The model hypothesized that CY P3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a g ood fit with the sigmoidal kinetic observations. Therefore, four pairs of t he kinetic estimates (K-S1, K-alpha, K-S2, k(beta), K-S3, k(delta), K-S4, a nd k(gamma)) were resolved to interpret the features of binding affinity an d catalytic ability of CYP3A4. Dissociation constants K-S1 and K-S2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than K-S3 and K-S4 for a two-substrate-bound enzyme (SES), while respectiv e rate constants k(delta) and k(gamma) were 3-218-fold greater than k(alpha ) and k(beta), implying that access and binding of the first molecule to ei ther site in an active pocket of CYP3A4 can enhance the binding affinity an d reaction rate of the vacant site for the second substrate. Thus our resul ts provide some new insights into the co-operative binding of two substrate s in the inner portions of an allosteric CYP3A4 active site.