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 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 H-1-NMR spectra identified the enantiomeric pairs. H-1-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)-alpha-methylbenzylamine, determined that the most inhibitory isomer is either 1S, 3R or 1R, 3S. The chemical shifts of protons in the thiolane I-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 Angstrom resolution of the ternary enzyme complexes with NADH demonstrated that the absolute configuration of the most inhibitory (K-ii = 0.31 mu M) stereoisomer is 1S, 3R and the next best inhibitor (K-ii = 0.73 mu M) 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.