Relationship between protein structure and methionine oxidation in recombinant human α1-antitrypsin
Relationship between protein structure and methionine oxidation in recombinant human α1-antitrypsin
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DOI：
10.1021/bi025599p
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发表时间：
2002-05-21
期刊：
影响因子：
2.9
通讯作者：
Cooney, CL
中科院分区：
文献类型：
--
作者：
Griffiths, SW;Cooney, CL
α 1-抗胰蛋白酶是一种亚稳态和构象柔性蛋白，属于蛋白酶抑制剂的丝氨酸蛋白酶抑制剂家族。尽管已知蛋白质活性位点的甲硫氨酸氧化导致生物活性的丧失，但关于蛋白质的每个甲硫氨酸残基的反应性的具体知识很少。在这项研究中，我们已经使用肽图谱研究的氧化动力学的α 1-抗胰蛋白酶的甲硫氨酸在α 1-AT（（C232 S））以及M351 L和M358 V突变体。这些动力学研究证实，Met 1、Met 226、Met 242、Met 351和Met 358在中性pH值下与过氧化氢反应，并且每种反应性蛋氨酸以双分子机制而非偶联机制氧化。Met 226，Met 351和Met 358氧化的分析提供了关于α 1-抗胰蛋白酶活性位点的结构的见解，使我们能够将构象与实验观察到的反应性联系起来。还检查了溶液pH值与蛋氨酸氧化之间的关系，以评价在干扰天然结构的条件下蛋氨酸的反应性。甲硫氨酸氧化数据表明，在pH 5时，发生全局构象变化，改变每个α 1-抗胰蛋白酶的10个甲硫氨酸残基的氧化敏感性。然而，在pH 6和9之间，发生更多局部构象变化，主要影响Met 242的反应性。总之，这项工作提供了一个详细的分析甲硫氨酸氧化α 1-抗胰蛋白酶，并提供了新的见解蛋白质的溶液结构。
alpha1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha1-antitrypsin's methionines in alpha1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha1-antitrypsin and offers new insights into the protein's solution structure.