The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro.
Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.
Men
Lijie
L
Department of Chemistry-027, University of California, Riverside, California 92521-0403, USA.
Wang
Yinsheng
Y
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D004220:Disulfides; D005656:Fungal Proteins; D010455:Peptides; D013438:Sulfhydryl Compounds; D013451:Sulfonic Acids; D006861:Hydrogen Peroxide; D000426:Alcohol Dehydrogenase; D015032:Zinc; D003545:Cysteine; D010100:Oxygen