Studies on the cosubstrate site of protease solubilized NADPH-cytochrome P450 reductase.
Modification of the protease solubilized NADPH-cytochrome P450 reductase (= NADPH-cytochrome c reductase) at the critical SH group in the cosubstrate binding site affects KmNADPH but not V for the cytochrome c reduction. The increase of KmNADPH is dependent on the size and the charge of the substituent introduced. Substitution of the cosubstrate site SH by the CN-, S2O3- and the (N-ethyl) succinimido group effects a 3-, 7- and 23-fold increase of KmNADPH, respectively. The critical SH group in the NADPH binding region can be specifically radiolabeled by N-ethyl (2,3-14C) maleimide after preincubation of the reductase with unlabeled NEM in the presence of 1 mM NADP+. The selective reaction at the essential cysteine in the cosubstrate site is demonstrated by peptide mapping of the thermolytic digest and urea SDS gel electrophoresis of the cyanogen bromide fragments of the reductase. Protease solubilized NADPH-cytochrome P450 reductase is inactivated by reagents directed to histidine, arginine and lysine residues. NADP (H) (1 mM) and 2'-AMP (1 mM) give effective protection only for the reaction of 1,2-cyclohexanedione (12 mM). The functional role of the basic amino acid residues for the cosubstrate binding by the NADPH-cytochrome P450 reductase cannot be established therefore by the modification experiments described. The number of NADPH binding sites in the NADPH-cytochrome P450 reductase is determined to one site/mol reductase by titration of the enzyme with NADP+ monitored by CD-spectroscopy.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010443:Peptide Chain Termination, Translational; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D011487:Protein Conformation; D013552:Swine
D000596:Amino Acids; D010446:Peptide Fragments; D009251:NADPH-Ferrihemoprotein Reductase; D010447:Peptide Hydrolases