STUDIES ON THE COSUBSTRATE SITE OF PROTEASE SOLUBILIZED NADPH-CYTOCHROME P450 REDUCTASE |
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Authors: | LUDWIG LUMPER,FRIEDHELM BUSCH, EHERAZADE D
ELI ,J
RG HENNING,TIBOR LAZAR |
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Affiliation: | LUDWIG LUMPER,FRIEDHELM BUSCH,ŠEHERAZADE DŽELIĆ,JÖRG HENNING,TIBOR LAZAR |
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Abstract: | Modification of the protease solubilized NADPH-cytochrome P450 reductase (= NADPH-cytochrome c reductase) at the critical SH group in the cosubstrate binding site affects KNADPHm but not V for the cytochrome c reduction. The increase of KNADPHm is dependent on the size and the charge of the substituent introduced. Substitution of the cosubstrate site SH by the CN-, S2 O3- and the (N-ethyl) succinimido group effects a 3-, 7- and 23-fold increase of KNADPHm 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, orginine 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. |
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Keywords: | basic amino acid residues CD-spectroscopy cosubstrate binding site modification of a critical SH-group |
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