Membrane charge as effector of cytochrome P-450LM2 catalyzed reactions in reconstituted liposomes.
The phospholipid specificity of rabbit liver microsomal cytochrome P-450LM2 catalyzed hydroxylation reactions was examined in reconstituted phospholipid vesicles. An apparent linear relationship between the negative charge of the vesicles and the rate of P-450LM2-catalyzed O-dealkylation of p-nitroanisole or 7-ethoxycoumarin was obtained. The membrane charge-mediated increase in hydroxylation activities was found not to be due to (i) an altered lipid/water partition coefficient of the substrate, (ii) a change in the apparent Michaelis constant of P-450LM2 for the substrate, (iii) a different activation energy of the O-demethylation of p-nitroanisole, (iv) different spin states of P-450LM2 or (v) an altered secondary structure of this enzyme as monitored by circular dichroism. However, when the formation of the ferrous carbonyl complex of P-450LM2 was followed under aerobic or anaerobic conditions after the addition of NADPH to the vesicles, an increased negative charge of the membrane was accompanied by an increased reducibility of P-450LM2. A similar linear relationship between the reducibility of cytochrome b5 and the negative charge of the liposomes was also evident in membranes containing NADPH-cytochrome P-450 reductase and cytochrome b5. It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology*; D011817:Rabbits; D013329:Structure-Activity Relationship
D003374:Coumarins; D008081:Liposomes; D010743:Phospholipids; C017299:7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase