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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.
Highlights
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
Initial rate measurements for the oxidation of ethanol and reduction of acetaldehyde can be described by an equation of the form (Dalziel, 1975)
Where v0 is the initial velocity, e is the active site concentration, and primed 4 constants are used for ethanol oxidation and unprimed 4 for acetaldehyde reduction
Summary
All solutions were prepared in a pH 7.00 buffer system of KHZPO, and K,HPO, which was 30 mM in phosphate. Stock inhibitor solutions were prepared immediately prior to use by weighing them into a volumetric flask and diluting with buffer. Inhibition kinetics of ethanol oxidation by electronegative alcohols were obtained by systematically varying the inhibitor and ethanol concentrations while holding NAD’ constant. Some of the less potent inhibitors (methanol, ethylene glycol, 2-iodoethanol, and 2chloroethanol) were oxidized slowly by LADH These background rates were determined under identical conditions in the absence of ethanol, and their inhibition runs were corrected, accounting for the ethanol/inhibitor ratio. Comparative studies of the inhibition of ethanol oxidation were carried out by premixing LADH, NAD+, and pyrazole and adding this mixture to a buffered ethanol solution. NAD+ concentration available for reaction with the inhibitor (Theorell et al 1969) In such cases, stronger inhibition is observed if the LADH. Modeling of kinetic data was done on a VAX/VMS mainframe using BMDPAR (1988) of BMDP Statistical Software, Inc
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