Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

• The cytochrome P450 reaction cycle has several microscopic steps.
• Rapid-kinetics experiments permit the study of transient intermediates.
• Single-turnover experiments determine if product release is rate-limiting.
• A kinetic isotope effect is observed if CH bond breaking is rate-limiting.

All cytochrome P450 enzyme reactions involve a catalytic cycle with several discreet physical or chemical steps. This cycle ends with the formation of the reactive heme iron–oxygen complex, which oxygenates substrate. While the steps might be very similar for each P450 enzyme, the rates of each step varies tremendously for each enzyme and sometimes even for different reactions catalyzed by the same enzyme. For example, the rate-limiting step for most bacterial P450 enzymes, with turnover numbers over 1000 s−1, is the second electron transfer. In contrast, steroidogenic P450s from eukaryotes catalyze much slower reactions, with turnover numbers of ∼5–250 min−1; therefore, assumptions about kinetic properties for the mammalian P450 enzymes based on the bacterial enzymes are tenuous. In order to dissect the rates for individual steps, special techniques that isolate individual steps and/or single turnovers are required. This article will review the theoretical principles and practical considerations for several of these techniques, with illustrative published examples. The reader should gain an appreciation for the appropriate methods used to interrogate particular steps in the P450 reaction cycle.

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