tailieunhanh - Báo cáo khoa học: Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes Effects of charge and nonbilayer phase propensity of the membrane

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aro-matic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 puri®ed insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. | Eur. J. Biochem. 269 1799-1805 2002 FEBS 2002 doi PRIORITY PAPER Epoxidation of benzo a pyrene-7 8-dihydrodiol by human CYP1A1 in reconstituted membranes Effects of charge and nonbilayer phase propensity of the membrane Pyotr Kisselev1 Dieter Schwarz1 Karl-Ludwig Platt2 Wolf-Hagen Schunck3 and Ivar Roots1 Institute of Clinical Pharmacology University Medical Centrum Charite Humboldt University of Berlin Germany 2Institute of Toxicology University of Mainz Germany 3Max Delbrueck Centrum for Molecular Medicine Berlin Germany Human cytochrome P4501A1 CYP1A1 is one of the key enzymes in the bioactivation of environmental pollutants such as benzo a pyrene B a P and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purihed insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol-min-1-pmol-1 CYP1A1 of the enantiomeric promutagens - - and -trans-7 8-dihy-droxy-7 8-dihydro-B a P 7 8-diol to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore the ratio of r-7 t-8-dihydroxy-t-9 10-epoxy-7 8 9 10-tetrahydro-B a P DE2 to r-7 t-8-dihydroxy-c-9 10-epoxy-7 8 9 10-tetrahydro-B a P DE1 formed from - -7 8-diol was signihcantly increased by the introduction of anionic lipids but not by that of nonbilayer lipids. Thus charged lipids affect the stereoselectivity of the epoxidation by leading to the formation