Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase.
Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc. Initially we confirmed the presence of two zinc atoms per subunit. Incubation of the enzyme with increasing concentrations of dithiothreitol, a method for partial chelation, allowed first the reduction of four disulphide bridges per enzyme, but eventually was sufficient to chelate the structural zinc atom without having any effect on the zinc located in the active site. The enzyme activity was not affected but the enzyme became very sensitive to heat denaturation. Chelation by EDTA was also performed. Given its location at an external position in the globular protein, protected in each subunit by one disulphide bridge, the results establish that the second zinc atom present on each enzymic subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of yeast alcohol dehydrogenase. Recovery experiments were performed by incubation of the native enzyme, or the dithiothreitol-treated enzyme, with a small amount of Zn2+. A stabilization effect was found when the structural zinc was re-incorporated after its removal by dithiothreitol. In all cases a large increase in activity was also observed, which was much greater than that expected based on the amount of re-incorporated zinc atom, suggesting the re-activation of some inactive commercial enzyme which had lost some of its original catalytic zinc atoms.
Magonet
E
E
Laboratoire de Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium.
Hayen
P
P
Delforge
D
D
Delaive
E
E
Remacle
J
J
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D006361:Heating; D006736:Horses; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D002614:Chelating Agents; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004492:Edetic Acid; D000426:Alcohol Dehydrogenase; D015032:Zinc; D004229:Dithiothreitol