KINETIC-STUDIES ON A GENETICALLY-ENGINEERED FUSED ENZYME BETWEEN RAT CYTOCHROME P4501A1 AND YEAST NADPH-P450 REDUCTASE

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent K-m and V-max values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s(-1), respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s(-1) using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant k(on) for substrate binding to the fused enzyme was 25 mM(-1).s(-1), which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent K-m and V-max values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s(-1), respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s(-1), respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes. Thus, the apparent V-max value and the reduction rate constant of P4501A1 in AH22/pAMR2 were about 20 times larger than those in AH22/pAMC1 in proportion to the content of NADPH-P450 reductase in the microsomal fractions. These results together with the spectral data for the hydroxylation reaction suggest that the rate-limiting step of the reaction by P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes is the first electron transfer from NADPH-P450 reductase to P4501A1.