Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.
Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide.
Tkachenko
A G
AG
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Winston
G W
GW
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D015003:Yeasts / Q000201:enzymology; D015032:Zinc / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000378:metabolism*
D013439:Sulfhydryl Reagents; D000431:Ethanol; D000249:Adenosine Monophosphate; D020122:tert-Butylhydroperoxide; D000426:Alcohol Dehydrogenase; D005978:Glutathione; D015032:Zinc; D004229:Dithiothreitol