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J. Biol. Chem., Vol. 277, Issue 50, 48960-48964, December 13, 2002
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From the Graduate Center for Toxicology, University of Kentucky,
Lexington, Kentucky 40536-0305
NADPH-cytochrome P-450 reductase is the electron
transfer partner for the cytochromes P-450, heme oxygenase, and
squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high
selectivity for NADPH and uses NADH only poorly. Substitution of
tryptophan 677 with alanine has been shown to yield a 3-fold increase
in turnover with NADH, but profound inhibition by
NADP+ makes the enzyme unsuitable for in
vivo applications. In the present study site-directed mutagenesis
of amino acids in the 2'-phosphate-binding site of the NADPH domain,
coupled with the W677A substitution, was used to generate a reductase
that was able to use NADH efficiently without inhibition by
NADP+. Of 11 single, double, and triple mutant proteins,
two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold
increase in catalytic efficiency
(kcat/Km) with NADH.
Inhibition by NADP+ was reduced by up to 4 orders of
magnitude relative to the W677A protein and was equal to or less than
that of the wild-type reductase. Both proteins were 2-3-fold more
active than wild-type reductase with NADH in reconstitution assays with
cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant
cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/W677A
protein increased the sensitivity to dimethylnitrosamine by
~2-fold, suggesting that the ability to use NADH afforded a
significant advantage in this in vivo assay.
Modification of the Nucleotide Cofactor-binding Site of
Cytochrome P-450 Reductase to Enhance Turnover with NADH in
Vivo*
*
This work was funded by National Science Foundation Grant
MCB-9808636 and National Institutes of Health Training Grant ES-07266.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: College of Pharmacy,
University of Kentucky, Lexington, KY 40536-0082. Tel.:
859-257-1137; Fax: 859-257-7564; E-mail: tporter@uky.edu.
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