A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy ω product formation

A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis–Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme–substrate (ES) complex responsible for ω hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to ω-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of ω to ω-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3 is able to produce the ω alcohol, a product that arises from the high-energy primary radical.