Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine.

Abstract

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)