Flexibility of Liver Alcohol Dehydrogenase in Stereoselective Binding of 3-Butylthiolane 1-Oxides

Thiolane 1-oxides are analogs of the carbonyl substrates that bind to the alcohol dehydrogenase−NADH complex and are potent uncompetitive inhibitors against alcohol [Chadha, V. K., et al. (1985) J. Med. Chem. 28, 36−40]. The four stereoisomers of 3-butylthiolane 1-oxide (BTO) were separated by chiral phase chromatography. CD and 1H-NMR spectra identified the enantiomeric pairs. 1H-NMR chemical shifts were assigned on the basis of COSY spectra of both diastereoisomers and confirmed by HMQC spectra. Coupling constants were determined through one-dimensional decoupling experiments. NMR with chiral shift reagents, Eu(hfc)3 [europium tris[3-[(heptafluoropropyl)hydroxymethylene]-(+)-camphorate]] or (R)-(−)-N-(3,5-dinitrobenzoyl)-α-methylbenzylamine, determined that the most inhibitory isomer is either 1S,3R or 1R,3S. The chemical shifts of protons in the thiolane 1-oxide ring were influenced by the whole structure and were not correlated with the computed Mulliken charges. X-ray crystallography at 2.1 and 1.66 Å resolution of the ternary enzyme complexes with NADH demonstrated that the absolute configuration of the most inhibitory (Kii = 0.31 μM) stereoisomer is 1S,3R and the next best inhibitor (Kii = 0.73 μM) is 1S,3S. The thiolane 1-oxide rings bind in the same position, in the substrate binding site, but the geometry of the complexes suggests that the sulfoxides are not transition state analogs. Significantly, the butyl groups of the two isomers are accommodated differently by flexible amino acid side chains adopting alternative rotameric conformations

Yeast Alcohol Dehydrogenase Structure and Catalysis

Yeast (<i>Saccharomyces cerevisiae</i>) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure