Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme.
Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.
D000345:Affinity Labels / Q000494:pharmacology; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D006736:Horses; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation
D000345:Affinity Labels; D007093:Imidazoles; C000314:alpha-bromo-beta-(5-imidazolyl)propionic acid; C006997:alpha-chloro-beta-imidazol-4(5)-ylpropionic acid; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase