Active site modifications in a double mutant of liver alcohol dehydrogenase: structural studies of two enzyme-ligand complexes

Biochemistry
T D ColbyB M Goldstein

Abstract

The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogenase (LADH) requires the transfer of a hydride ion from the alcohol substrate to the cofactor nicotinamide adenine dinucleotide (NAD). A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated in a number of LADH mutants designed to enhance or diminish this effect [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. The active site double mutant Phe93 --> Trp/Val203 --> Ala shows a 75-fold reduction in catalytic efficiency relative to that of the native enzyme, and reduced tunneling relative to that of either single mutant. We present here two crystal structures of the double mutant: a 2.0 A complex with NAD and the substrate analogue trifluoroethanol and a 2.6 A complex with the isosteric NAD analogue CPAD and ethanol. Changes at the active site observed in both complexes are consistent with reduced activity and tunneling. The NAD-trifluoroethanol complex crystallizes in the closed conformation characteristic of the active enzyme. However, the NAD nicotinamide ring rotates away from the substrate, toward the space vacated by replacement of Val203 with the smaller alanine. Replacement of Phe93 wit...Continue Reading

References

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Citations

Jun 1, 2000·Advances in Enzyme Regulation·B M Goldstein, T D Colby
Jun 25, 1999·Chemistry & Biology·A Kohen, J P Klinman
Dec 17, 2011·Journal of the American Chemical Society·Vanja StojkovićAmnon Kohen
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Nov 8, 2019·The Journal of Physical Chemistry. B·Chethya RanasingheChristopher M Cheatum

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