title: Ionization-linked cooperative interactions in the active site of horse liver alcohol dehydrogenase.
abstract: Affinity labeling of horse liver alcohol dehydrogenase with iodoacetate in the presence of the activator imidazole has been studied from pH 6.1 to 10.5. The pH profiles for the dissociation constants of iodoacetate from the free enzyme and the enzyme-imidazole complex and of imidazole from the free enzyme and the binary enzyme-iodoacetate complex were determined. The variation with pH of the dissociation constants of iodoacetate (KI) and imidazole (KL) have in common a pKa of 8.6 assigned to the zinc-water ionization, and a pKa near 10. Lysine modification by ethyl acetimidate results in a higher affinity of iodoacetate to the enzyme at high pH as the pKa values of the lysine residues are increased. The binding of iodoacetate and imidazole at each enzyme subunit shows negative cooperativity at pH less than 9, with an interaction constant of 4.8 at pH 6.1. Positive cooperativity is observed at pH greater than 9, with an interaction constant of 0.5 at pH 10.5. The pH-dependent change in cooperativity results from the removal of the zinc-water ionization when imidazole becomes coordinated to the catalytic zinc ion. When iodoacetate binds at the anion binding site, a large perturbation of the zinc-water ionization is observed. Unlike imidazole, the binding of 1,10-orthophenanthroline and iodoacetate shows positive cooperativity at both pH 8.2 and 10.0 with an interaction constant as low as 0.06 at pH 10.0.
mesh_terms: D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D015032:Zinc
chemical_list: D007093:Imidazoles; D007461:Iodoacetates; C029899:imidazole; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D015032:Zinc