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J. Biol. Chem., Vol. 266, Issue 30, 19976-19980, 10, 1991
AL Shen, MJ Christensen and CB Kasper
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.
NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
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