STEREOSELECTIVE AFFINITY LABELING OF SHEEP LIVER SORBITOL DEHYDROGENASE BY CHLOROSUBSTITUTED ANALOGS OF 2-BROMO-3-(5-IMIDAZOLYL) PROPIONIC-ACID

The role of configuration for the affinity labelling of sheep liver so rbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-i midazolyl)propionate (BrImPpOH) has been studied. A saturation kinetic s mechanism applies which includes formation of a reversible complex w ith the enzyme prior to alkylation of Cys-43. The pseudo first-order i nactivation rate-constant, k(2), and the dissociation constant for the reversible enzyme-affinity label complex, K-EI, were determined at pH 7.4 and 23.5 degrees C. The stereo isomers of each affinity label exh ibit different kinetic characteristics but, unlike with horse liver al cohol dehydrogenase, the discrimination between them is not absolute. For the different affinity labels, k(2) varies with 2-chloro-3-(5-imid azolyl)methylpropionat (Me-ClImPpOH)> 2-chloro-3-(5-imidazolyl)propion ate (ClImPpOH)> 2-chloro-3-(5-imidazolyl)propanol (ClImPOH), consisten t with their order of inherent reactivity, and the specificity constan t k(2)/K-EI varies with (S)-Me-ClImPpOH > (S)-ClImPpOH > (S)-ClImPOH > (R)-Me-ClImPpOH > (R)-ClImPpOH. Models of the affinity labels were bu ilt into the active site of the predicted subunit structure of the enz yme by using a computer-controlled display system. In each binary comp lex, the imidazole moiety of the affinity label was liganded to the ca talytic zinc atom, and the angle S-cys-C-alpha-Cl was linear, in accor dance with an S(N)2 mechanism. Both enantiomers of each label could fo rm plausible complexes with the enzyme model, in agreement with the ki netic data. The enantiomeric selectivity, rather than absolute specifi city, of the reaction appears due to the anion-binding site in sorbito l dehydrogenase being less developed than in horse liver alcohol dehyd rogenase.