title: 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)
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: W G
authors.initials: WG
authors.lastname: Balvers
authors.forename: M G
authors.initials: MG
authors.lastname: Boersma
authors.forename: C
authors.initials: C
authors.lastname: Veeger
authors.forename: I M
authors.initials: IM
authors.lastname: Rietjens
mesh_terms: D000818:Animals; D003374:Coumarins / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003640:Dealkylation; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D003374:Coumarins; D010078:Oxazines; D010713:Phosphatidylcholines; C015153:1,2-dilauroylphosphatidylcholine; C017299:7-ethoxycoumarin; C007649:ethoxyresorufin; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C010942:1,2-distearoyllecithin; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1