Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution.
The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis. This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme. Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues. Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity. It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom. In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG. The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Rescigno
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Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, United Kingdom.
Perham
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D003903:Deuterium; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D009249:NADP; D003903:Deuterium; D005980:Glutathione Reductase; D005978:Glutathione; D019803:Glutathione Disulfide