1A72

AN ACTIVE-SITE DOUBLE MUTANT (PHE93->TRP, VAL203->ALA) OF HORSE LIVER ALCOHOL DEHYDROGENASE IN COMPLEX WITH THE ISOSTERIC NAD ANALOG CPAD

Summary for 1A72

• Entry DOI: 10.2210/pdb1a72/pdb
• Descriptor: HORSE LIVER ALCOHOL DEHYDROGENASE, ZINC ION, 5-BETA-D-RIBOFURANOSYLPICOLINAMIDE ADENINE-DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: oxidoreductase (nad(a)-choh(d)), active site mutant, liver alcohol dehydrogenase, isosteric nad inhibitors, oxidoreductase
• Biological source: Equus caballus (horse)
• Cellular location: Cytoplasm: P00327
• Total number of polymer chains: 1
• Total formula weight: 40658.50

Authors

Colby, T.D.,Bahnson, B.J.,Chin, J.K.,Klinman, J.P.,Goldstein, B.M. (deposition date: 1998-03-19, release date: 1998-06-17, Last modification date: 2024-05-22)

Primary citation

Colby, T.D.,Bahnson, B.J.,Chin, J.K.,Klinman, J.P.,Goldstein, B.M.
Active site modifications in a double mutant of liver alcohol dehydrogenase: structural studies of two enzyme-ligand complexes.
Biochemistry, 37:9295-9304, 1998

PubMed 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 with the larger tryptophan also produces unfavorable steric contacts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These contacts are relieved in the second complex by rotation of the CPAD pyridine ring into an unusual syn orientation. The resulting loss of the carboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme.

PubMed: 9649310
DOI: 10.1021/bi973184b

Experimental method

X-RAY DIFFRACTION (2.6 Å)