Stereo-selective affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionic acid.

The role of configuration for the affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionate (BrImPpOH) has been studied. A saturation kinetics mechanism applies which includes formation of a reversible complex with the enzyme prior to alkylation of Cys-43. The pseudo first-order inactivation rate-constant, k2, and the dissociation constant for the reversible enzyme-affinity label complex. KEI, were determined at pH 7.4 and 23.5 degrees C. The stereo isomers of each affinity label exhibit different kinetic characteristics but, unlike with horse liver alcohol dehydrogenase, the discrimination between them is not absolute. For the different affinity labels, k2 varies with 2-chloro-3-(5-imidazolyl)methylpropionate (Me-ClImPpOH) > 2-chloro-3-(5-imidazolyl)propionate (ClImPpOH) > 2-chloro-3-(5-imidazolyl)propanol (ClImPOH), consistent with their order of inherent reactivity, and the specificity constant k2/KEI varies with (S)-Me-ClImPpOH > (S)-ClImPpOH > (S)-ClImPpOH > (R)-Me-ClImPpOH > (R)-ClImPpOH. Models of the affinity labels were built into the active site of the predicted subunit structure of the enzyme by using a computer-controlled display system. In each binary complex, the imidazole moiety of the affinity label was liganded to the catalytic zinc atom, and the angle Scys-C alpha-Cl was linear, in accordance with an SN2 mechanism. Both enantiomers of each label could form plausible complexes with the enzyme model, in agreement with the kinetic data. The enantiomeric selectivity, rather than absolute specificity, of the reaction appears due to the anion-binding site in sorbitol dehydrogenase being less developed than in horse liver alcohol dehydrogenase.