Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.
Abstract: 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.
Copyright 2000 Academic Press.
Publication Date: 2000-07-20 PubMed ID: 10900146DOI: 10.1006/abbi.2000.1912Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research article examines how an oxidative agent, tert-butyl hydroperoxide (tBOOH), affects the activity of the alcohol dehydrogenase enzyme (ADH) in horse liver (HLADH) and yeast (YADH). The study found that tBOOH elevates the performance of HLADH while inhibiting that of YADH.
Stimulation of Horse Liver Alcohol Dehydrogenase
- The researchers found that when they allowed HLADH to interact with tBOOH before observation, the ADH’s activity doubled. This was measurable as an increase in the enzyme’s ability to oxidize ethanol.
- This tBOOH-induced stimulation was concentration-dependent up to a specific point (100 mM) after which no additional stimulation was observed.
- To understand the mechanism behind this elevated activity, the researchers used various reagents that are known to interact with the ADH enzyme. This allowed specific elements of the enzyme’s structure to be isolated and their role in the stimulation observed.
- The researchers discovered that complexes with NAD(+) or NADH, which bind to Arg-47 and Arg-369, were able to reduce the rate and extent of the stimulation caused by tBOOH.
- However, AMP or sulfhydryl reagents did not prevent this tBOOH-induced stimulation, hinting that Lys-228 and sulfhydryl group oxidation had no part in the process.
Inhibition of Yeast Alcohol Dehydrogenase
- In contrast to the stimulating effect observed in HLADH, YADH was inhibited irreversibly by tBOOH. The degree of this inhibition increased at a steady rate as higher concentrations of tBOOH were used.
- This inhibition was associated with the alteration of four -SH groups per YADH molecule, whereas only two were modified in the case of HLADH.
Underlying Mechanisms
- The researchers suggest that the stimulation of HLADH by tBOOH may be due to a destabilizing effect on the enzyme’s catalytic Zn-coordination sphere and those amino acids linked to the enzyme’s co-factor binding in the active site.
- Conversely, the inhibition of YADH by tBOOH was thought to be linked to the oxidation of -SH groups by the oxidative agent.
Cite This Article
APA
Tkachenko AG, Winston GW.
(2000).
Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.
Arch Biochem Biophys, 380(1), 165-173.
https://doi.org/10.1006/abbi.2000.1912 Publication
Researcher Affiliations
- Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
MeSH Terms
- Adenosine Monophosphate / pharmacology
- Alcohol Dehydrogenase / metabolism
- Animals
- Binding Sites
- Dithiothreitol / pharmacology
- Dose-Response Relationship, Drug
- Ethanol / pharmacology
- Glutathione / pharmacology
- Horses
- Hydrogen-Ion Concentration
- Kinetics
- Liver / enzymology
- Protein Binding
- Sulfhydryl Reagents / pharmacology
- Time Factors
- Yeasts / enzymology
- Zinc / metabolism
- tert-Butylhydroperoxide / metabolism
Grant Funding
- AA06758 / NIAAA NIH HHS
Citations
This article has been cited 2 times.- Jin L, Szeto KY, Zhang L, Du W, Sun H. Inhibition of alcohol dehydrogenase by bismuth. J Inorg Biochem 2004 Aug;98(8):1331-7.
- Berrada W, Naya A, Iddar A, Bourhim N. Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis). Mol Cell Biochem 2002 Feb;231(1-2):117-27.
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