title: Involvement of histidine residues in the activity of horse liver alcohol dehydrogenase.
abstract: X-ray crystallographic studies indicate that His-51 in alcohol dehydrogenase may participate in a proton relay system during enzymatic catalysis [Eklund, H., Plapp, B. V., Samama, J.-P., & Brändén, C.-I. (1982) J. Biol. Chem. 257, 14349-14358], but there is no direct chemical evidence for this role. Diethyl pyrocarbonate (0.5-2 mM, pH 8, 25 degrees C) rapidly inactivated alcohol dehydrogenase, which was acetimidylated on all accessible lysine residues in order to prevent their modification. The reaction appeared to be specific for histidine residues, and the enzyme could be reactivated with 0.5 M hydroxylamine. The ethoxyformylated enzyme could still bind coenzymes, substrate analogues, and bipyridine, but with decreased affinity. The relationship between enzyme activity and the number of histidine residues modified showed that two histidine residues are modified during inactivation. NADH and isobutyramide significantly reduced the rate of inactivation, and the loss of activity then correlated with the modification of one to two histidine residues. The pH dependence of the inactivation showed the unusually high pK value of 9.6, which we attribute to the ionization of the water bound to zinc in the proton relay system. Although the histidine residue involved in the inactivation has not been identified, we conclude that one histidine residue (probably His-51) is essential for enzymatic activity.
mesh_terms: D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D001051:Apoenzymes; D009243:NAD; D006639:Histidine; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D004047:Diethyl Pyrocarbonate