ZINC-ACTIVATED ALCOHOLS IN TERNARY COMPLEXES OF LIVER ALCOHOL-DEHYDROGENASE

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POCKER, Y
PAGE, JD
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Q5 [生物化学]; Q7 [分子生物学];
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071010 ; 081704 ;
摘要
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 pK(a) 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 pK(a) can be derived to show that the pK(a) of an alcohol bound in a ternary complex is also dependent on its pK(a) as a free alcohol. Ternary complex pK(a) values have been determined for ethanol and the inhibiting alcohols.
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页码:22101 / 22108
页数:8
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