Kinetic Evidence for an Induced-Fit Mechanism in the Binding of the Substrate Camphor by Cytochrome P450cam

Bacterial cytochrome P450 (P450) 101A1 (P450cam) has served as a prototype among the P450 enzymes and has high catalytic activity toward its cognate substrate, camphor. X-ray crystallography and NMR and infrared (IR) spectroscopy have demonstrated the existence of multiple conformations of many P450s, including P450cam. Kinetic studies have indicated that substrate binding to several P450s is dominated by a conformational selection process, in which the substrate binds an individual conformer(s) of the unliganded enzyme. P450cam was found to differ in that binding of the substrate camphor is dominated by an induced-fit mechanism, in which the enzyme binds camphor and then changes conformation, as evidenced by the equivalence of binding eigenvalues observed when varying both camphor and P450cam concentrations. The accessory protein putidaredoxin had no effect on substrate binding. Estimation of the rate of dissociation of the P450cam·camphor complex (15 s–1) and fitting of the data yield a minimal kinetic mechanism in which camphor binds (1.5 × 107 M–1 s–1) and the initial P450cam·camphor complex undergoes a reversible equilibrium (kforward 112 s–1, kreverse 28 s–1) to a final complex. This induced-fit mechanism differs from those reported for several mammalian P450s and bacterial P450BM-3, indicative of the diversity of how P450s recognize multiple substrates. However, similar behavior was not observed with the alternate substrates (+)-α-pinene and 2-adamantanone, which probably utilize a conformational selection process.