Structure of the NADPH-binding motif of glutathione reductase: Efficiency determined by evolution

Maria Rescigno, Richard N. Perham

Research output: Contribution to journalArticlepeer-review

Abstract

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 α-helix in the βαβ 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 K m 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 K m 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. The marked preference for a glycine residue at the N-terminus of the α-helix in the βαβ fold revealed by these experiments is in keeping with the view that the lack of a side chain at this position facilitates an electrostatic interaction between the dipole positive charge of the helix and the negative charge of the pyrophosphate bridge of the bound coenzyme. There was no evidence of a significant conformational change in any of the mutant proteins. Replacement of this glycine residue by alanine should act, therefore, as a safe diagnostic test for the involvement of a putative fingerprint motif in the binding of a dinucleotide by an unknown protein.

Original languageEnglish
Pages (from-to)5721-5727
Number of pages7
JournalBiochemistry
Volume33
Issue number19
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry

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