The Oxidation of Yeast Alcohol Dehydrogenase-1 by Hydrogen Peroxide in Vitro

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• 作者：Lijie Men ; Yinsheng Wang
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：January 2007
• 年：2007
• 卷：6
• 期：1
• 页码：216 - 225
• 全文大小：280K
• 年卷期：v.6,no.1(January 2007)
• ISSN：1535-3907

文摘

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydesor ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity ingrowing 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/MSanalysis 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 theactive site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO₂H) and cysteinesulfonic 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 ofproteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO₂H and Cys-SO₃His also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitativemeasurement results revealed that, among all the cysteine residues, Cys⁴³ was the most susceptibleto 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.