Abstract

Steady-state kinetic and deuterium isotope effect studies have been conducted to determine the influence of the phospholipid dilauroylphosphatidylcholine on the catalytic activity of a reconstituted monooxygenase system composed of cytochrome P-450 and NADPH-cytochrome c ( P-450) reductase. Addition of this lipid up to a concentration equivalent to its CMC resulted in an increase in V for benzphetamine N-demethylation. Above the CMC, no further change in V was observed. In contrast, the K m was not affected throughout the entire lipid concentration range. Furthermore, the deuterium isotope effect for 7-ethoxycoumarin O-deethylation was not affected by the lipid concentration indicating that the contribution of the carbon-hydrogen bond cleavage step to V was also not affected. These data are consistent with the mass-action model proposed earlier (G. T. Miwa, S. B. West, M. T. Huang, and A. Y. H. Lu, (1979), J. Biol. Chem. 254, 5695–5700) for cytochrome P-450 and NADPH-cytochrome c ( P-450) reductase association during catalysis. The lipid concentration, below its CMC, appears to decrease the apparent dissociation constant for the cytochrome P-450-reductase complex thus causing an increase in the steady-state concentration of this catalytically active complex.

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