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J. Biol. Chem., Vol. 277, Issue 50, 48960-48964, December 13, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/50/48960    most recent M210173200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Elmore, C. L. Articles by Porter, T. D. Search for Related Content PubMed PubMed Citation Articles by Elmore, C. L. Articles by Porter, T. D. Social Bookmarking                      What's this?

Modification of the Nucleotide Cofactor-binding Site of Cytochrome P-450 Reductase to Enhance Turnover with NADH in Vivo^*

C. Lee Elmore and Todd D. Porter

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 (k_cat/K_m) 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.

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