title: Zinc-activated alcohols in ternary complexes of liver alcohol dehydrogenase.
abstract: Activation parameters for each reaction step in the kinetic mechanism of liver alcohol dehydrogenase have been measured for the oxidation of ethanol and the reduction of acetaldehyde. In the oxidation process, the highest enthalpy of activation, 9.7 kcal/mol, occurs for the turnover of the liver alcohol dehydrogenase-NAD(+)-ethanol ternary complex. To investigate if this enthalpy requirement represents a change in the ionization state of ethanol bound in the ternary complex, inhibition of ethanol oxidation was determined using the following series of small, electronegative alcohols with pKa values ranging from 12.37 to 15.5: 2,2,2-trifluoroethanol, 2,2,2-trichloroethanol, 2,2,2-tribromoethanol, 2,2-dichloroethanol, 2,2-difluoroethanol, propargyl alcohol, 3-hydroxypropionitrile, 2-chloroethanol, 2-iodoethanol, 2-methoxyethanol, ethylene glycol, and methanol. The observed inhibition patterns were analyzed according to several kinetic inhibition models; in each case, the best fit model was used to determine the substrate competitive inhibition constant. A plot of the logarithm of these inhibition constants is shown to be dependent on the pKa values of the inhibiting alcohols with a slope approaching -1, indicating that inhibition is controlled by a proton loss from the alcohol. The observed competitive inhibition behavior, coupled with crystallographic studies depicting a direct ligation of an alcohol oxygen to the catalytic zinc ion, indicates that inhibition is controlled by the formation of a zinc-bound alkoxide. Because the inhibiting alcohols are structurally homologous to ethanol, a relationship between the inhibition constant and the inhibiting alcohol's pKa can be derived to show that the pKa of an alcohol bound in a ternary complex is also dependent on its pKa as a free alcohol. Ternary complex pKa values have been determined for ethanol and the inhibiting alcohols.
authors: Department of Chemistry, University of Washington, Seattle 98195.
authors: Y
authors: Y
authors: Pocker
authors: J D
authors: JD
authors: Page
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism*; D000818:Animals; D000431:Ethanol / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D000432:Methanol / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics; D015032:Zinc / Q000494:pharmacology*
chemical_list: D000438:Alcohols; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D015032:Zinc; D000432:Methanol