Rabbit liver cytochrome P-450 LM2: roles of substrates, inhibitors, and cytochrome b5 in modulating the partition between productive and abortive mechanisms.

Abstract

Cytochrome b5 (b5) enhanced the rate of 7-ethoxycoumarin deethylation by rabbit liver cytochrome P-450 LM2. The effect was saturable and can be analyzed as the sum of two effects: a decrease in the KM for the substrate and an increase in the Vmax. When two substrates were present simultaneously, they competed in a complex way depending on the presence of b5. Various substrates at low concentrations inhibited 7-ethoxycoumarin deethylation in a competitive-like way. Only a part of the P-450 activity was found to be affected by this mode of inhibition. Higher inhibitor concentrations caused a new kind of inhibition characterized by much higher half-effect values. The pattern seemed dependent on the ability of the inhibitors to be metabolized and was dramatically changed by the addition of b5. The relative rates of P-450-dependent NADPH oxidation and hydrogen peroxide and water formation were determined as well as their dependence on substrate and b5. A steady-state kinetic model that includes two branch points for water, hydrogen peroxide, and product formation is proposed. The model allows a full prediction of the b5 effects and seems consistent with most of the steady-state and rapid kinetic data available in the literature.