NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding.
Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
A L
AL
Shen
M J
MJ
Christensen
C B
CB
Kasper
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D009251:NADPH-Ferrihemoprotein Reductase; D003545:Cysteine