ACTIVE-SITE MODIFICATIONS IN A DOUBLE MUTANT OF LIVER ALCOHOL-DEHYDROGENASE - STRUCTURAL STUDIES OF 2 ENZYME-LIGAND COMPLEXES

The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogen ase (LADH) requires the transfer of a hydride ion from the alcohol sub strate to the cofactor nicotinamide adenine dinucleotide (NAD). A quan tum mechanical tunneling contribution to this hydride transfer step ha s 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 Phe(93) - -> Trp/Val(203) --> Ala shows a 75-fold reduction in catalytic efficie ncy relative to that of the native enzyme, and reduced tunneling relat ive to that of either single mutant. We present hen two crystal struct ures of the double mutant: a 2.0 Angstrom complex with NAD and the sub strate analogue trifluoroethanol and a 2.6 Angstrom complex with the i sosteric NAD analogue CPAD and ethanol. Changes at the active site obs erved in both complexes are consistent with reduced activity and tunne ling. The NAD-trifluoroethanol complex crystallizes in the closed conf ormation characteristic of the active enzyme. However, the NAD nicotin amide ring rotates away from the substrate, toward the space vacated b y replacement of Val(203) with the smaller alanine. Replacement of Phe (93) with the larger tryptophan also produces unfavorable steric conta cts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These co ntacts are relieved in the second complex by rotation of the CPAD pyri dine ring into an unusual syn orientation. The resulting loss of the c arboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme.