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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Oct 1;88(19):8420–8424. doi: 10.1073/pnas.88.19.8420

Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

E G Weinhold 1, A Glasfeld 1, A D Ellington 1, S A Benner 1
PMCID: PMC52520  PMID: 1924300

Abstract

Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its kcat value and makes one stereochemical "mistake" every 850,000 turnovers (instead of approximately 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a cofactor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochemical "mistake" every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochemical fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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