The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase.
Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Strobel
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HW
D000089:Acetic Anhydrides / Q000494:pharmacology; D000818:Animals; D001555:Benzene Derivatives / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D008239:Lysine / Q000737:chemistry*; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D000089:Acetic Anhydrides; D001555:Benzene Derivatives; D003374:Coumarins; C031800:acetic anhydride; C017299:7-ethoxycoumarin; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D008239:Lysine; C007164:cumene hydroperoxide