Kinetics of cytochromes P‐450 IA1 and IIB1 in reconstituted systems with dilauroyl‐ and distearoyl‐glycerophosphocholine
Authors: BALVERS Walter G. , BOERSMA Marelle G. , VEEGER Cees , RIETJENS Ivonne M. C. M. 
Published: March 2005

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 ethoxy‐coumarin 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‐α‐dilauroyl‐sn‐glycero‐3‐phosphocholine (Lau2GroPCho) system compared with the l‐α‐distearoyl‐sn‐glycero‐3‐phosphocholine (Ste2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Ksm) 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‐IA1‐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 Ksm and the V.In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Ksm 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. With respect to the differential effect of Lau2GroPCho and Ste2GroPCho on the Ksm of P‐450 IA1 and IIB1 the results demonstrate that this phenomenon does not result from a differential effect of the two acyl2GroPCho species on either the substrate binding, the oxygen binding or the rate of electron transfer. Therefore, it was concluded that the differential effect of Lau2GroPCho and Ste2GroPCho on the Ksm of P‐450 IA1 and IIB1 must result from an effect of the acyl2GroPCho on the kinetic parameters of other steps in the P‐450 cycle such as reductive oxygen splitting, substrate conversion and/or product release.