Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor.
Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.
D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010710:Phosphates / Q000378:metabolism*; D051381:Rats; D014302:Trinitrobenzenesulfonic Acid
D003067:Coenzymes; D003574:Cytochrome c Group; D010710:Phosphates; D014302:Trinitrobenzenesulfonic Acid; D004228:Dithionitrobenzoic Acid; D009251:NADPH-Ferrihemoprotein Reductase; D008239:Lysine; D003545:Cysteine