NADPH-cytochrome P-450 reductase (pig liver). Studies on the sequence of the cyanogen bromide peptides from the catalytic domain and on the reactivity of the thiol groups.
The reactivity of the cysteine residues in the non-denatured catalytic domain of the NADPH-cytochrome P-450 reductase (pig liver) was studied using the -SH reagent monobromobimane. Prerequisite was the characterization of the cysteine residues by their surrounding amino-acid sequences. In pursuit of these aims the CNBr fragments obtained from the catalytic domain were sequenced. The cysteine residues are distributed on six CNBr fragments of the catalytic domain [Vogel and Lumper (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1074]. Only the 11-kDa CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr- and Ala-Ser-Ser-Ser-, respectively, contain two cysteine residues each. The cysteine residues of the catalytic domain accessible to monobromobimane were localized on three CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser- and Ala-Arg-Asp-Val-, respectively. Inactivation of the trypsin-solubilized enzyme by -SH-directed reagents is caused by the modification of the accessible cysteine residue (which can be protected by NADPH) in the 11-kDa CNBr fragment (N-terminal sequence: Val-Gly-Pro-Thr-). The cosubstrate NADPH protected a second cysteine residue localized in the 11-kDa CNBr peptide with the N-terminal sequence Ala-Ser-Ser-Ser-, which is however modified at a distinctly slower rate than the critical cysteine residue characterized by the sequence -Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg-Arg. Five non-reacting thiol groups were localized on CNBr fragments with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser-, Ser-Leu-Asn-Asn-, Gly-Lys-Tyr-Val-Asp- and Ala-Ala-Asp-Pro-.
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments; D013329:Structure-Activity Relationship; D013552:Swine
D010446:Peptide Fragments; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase; D003545:Cysteine; D003488:Cyanogen Bromide