REVERSIBLE INHIBITION OF SHEEP LIVER SORBITOL DEHYDROGENASE BY THIOL COMPOUNDS

Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme-cat alyzed reaction competitively with respect to sorbitol, due to the for mation 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 i s indicated for some bifunctional thiols in their ternary complexes. E nzyme-bound thiolate facilitates NAD binding to the enzyme and vice ve rsa, mainly due to mutual electrostatic stabilization. The aromatic th iols 1-thio-1-phenylmethane and 1-thio-2-phenylethane are especially p otent inhibitors with an inhibition constant of 0.30 mu M at pH 9.9. T he inhibitory effect of aliphatic thiols, which is positively correlat ed with alkyl chain length, parallels that observed previously with th e related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibito r 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 pote nt sorbitol dehydrogenase inhibitors. The thionucleosides 6-thioguanos ine 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 inhi bitor specificities of the enzyme, the kinetic constants for several s orbitol dehydrogenase substrates were determined. L-threitol and DL-1- phenyl-1,2-ethanediol are good substrates with, at high pH, kinetic co nstants similar to those of sorbitol. The potent inhibition by dithiot hreitol and the aromatic thiols thus parallels the substrate specifici ty 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) an d 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 . 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 de hydrogenase active sites and confirms the activity assay of the enzyme .