Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme-catalyzed reaction competitively with respect to sorbitol, due to the formation of ternary enzyme-NAD-thiol complexes. The primary interaction of thiol inhibitors with the enzyme active site involves the catalytic zinc atom, and a bidentate mode of binding to the active site metal is indicated for some bifunctional thiols in their ternary complexes. Enzyme-bound thiolate facilitates NAD binding to the enzyme and vice versa, mainly due to mutual electrostatic stabilization. The aromatic thiols 1-thio-1-phenylmethane and 1-thio-2-phenylethane are especially potent inhibitors with an inhibition constant of 0.30 microM at pH 9.9. The inhibitory effect of aliphatic thiols, which is positively correlated with alkyl chain length, parallels that observed previously with the related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibitor binding. Several reversible inhibitors afford competitive protection against affinity labelling of the enzyme by 2-bromo-3-(5-imidazolyl) propionic acid due to the formation of binary enzyme-thiol complexes. The present study establishes thionucleosides as a novel class of potent sorbitol dehydrogenase inhibitors. The thionucleosides 6-thioguanosine and 6-thioinosine gave mixed inhibition with respect to sorbitol, due to the formation of enzyme-NAD-inhibitor and enzyme-NADH-inhibitor complexes. In order to enable a correlation of the substrate and inhibitor specificities of the enzyme, the kinetic constants for several sorbitol dehydrogenase substrates were determined. L-threitol and DL-1-phenyl-1,2-ethanediol are good substrates with, at high pH, kinetic constants similar to those of sorbitol. The potent inhibition by dithiothreitol and the aromatic thiols thus parallels the substrate specificity of the enzyme. The sorbitol competitive inhibitor 1-thiosorbitol is also a substrate with, at pH 7.4, a maximum velocity of 0.17 s-1 and a Michaelis constant of 8.6 mM. Dithiothreitol forms a tight ternary complex with the enzyme-NAD complex with a molar absorbance of 16.4 x 10(3) M-1 . cm-1 at 311 nm. A spectrophotometric titration of the enzyme with NAD in the presence of dithiothreitol is described, which enables an accurate determination of the concentration of sorbitol dehydrogenase active sites and confirms the activity assay of the enzyme.