Oxidation of Cys278 of ADH I isozyme from Kluyveromyces lactis by naturally occurring disulfides causes its reversible inactivation.
The inactivation of the homotetrameric cytosolic alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) by naturally occurring disulfides, oxidized glutathione, cystine and cystamine, was studied. The inactivation was fully reversed by dithiothreitol. The nicotinamide coenzyme, but not the substrate ethanol, protected KlADH I from inactivation. Gel filtration experiments and SDS-PAGE analysis, also, revealed that enzyme inactivation coincides with inter-subunits disulfide bond formation which are noticeably enhanced after prolonged oxidation with GSSG. Moreover, oxidized KlADH I, as its reduced state, retained the tetrameric stucture and appears mainly as a dimer under non-reducing SDS-PAGE. Conversely, KlADH I Cys278Ile mutant is unaffected by disulfides treatment. Therefore, in vitro, KlADH I wild-type could exist in two reversible forms: reduced (active) and oxidized (inactive), in which the Cys278 residues of each tetramer are linked by disulfide bonds. The redox state of KlADH I could represent the path for modulating its activity and then a regulatory step of glycolysis under hypoxic conditions. It might be hypothesized that KlADH I could represent an important target in redox signaling of Kluyveromyces lactis cell by inhibiting, under oxidative stress, the glycolytic pathway in favor of the pentose-phosphate shunt to restore its reducing potential.
Bucciarelli
Tonino
T
Department of Biomedical Sciences, University of Chieti "G. D'Annunzio", I-66013 Chieti, Italy.
Saliola
Michele
M
Brisdelli
Fabrizia
F
Bozzi
Argante
A
Falcone
Claudio
C
Di Ilio
Carmine
C
Martini
Filippo
F
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019803:Glutathione Disulfide / Q000737:chemistry; D007716:Kluyveromyces / Q000201:enzymology; D010084:Oxidation-Reduction; D016415:Sequence Alignment
D004220:Disulfides; D000426:Alcohol Dehydrogenase; D003545:Cysteine; D019803:Glutathione Disulfide