Transfer of oxygen from an artificial protease to peptide carbon during proteolysis.
Transfer of oxygen from an artificial protease to peptide carbon during proteolysis.
复制标题
在蛋白水解过程中，氧从人工蛋白酶转移到肽碳。
DOI：
10.1073/pnas.88.23.10578
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发表时间：
1991
影响因子：
11.1
通讯作者：
Meares,CF
中科院分区：
文献类型：
--
作者：
Rana,TM;Meares,CF
用金属螯合物对蛋白质进行位点特异性裂解是一种设计人工蛋白水解试剂的方法，该试剂是通过接近肽键而不是通过氨基酸残基类型来指导的。在抗坏血酸和 H2O2 存在的情况下，附着在人碳酸酐酶 I 的 Cys-212 上的铁螯合物快速裂解残基 Leu-189 和 Asp-190 之间的蛋白质，产生两个离散的片段。通过质谱分析证明了 18O 原子从 [18O]H2O2（或 [18O]O2）转移到 Leu-189 的羧基上。定量实验表明，一分子 H2O2 和一分子抗坏血酸可水解一个肽键（1:1:1 化学计量），并且该反应需要抗坏血酸和 H2O2。该过程是催化性的，因为对牛血清白蛋白的相关实验揭示了每个裂解的多肽链的两个裂解事件。羟基自由基清除剂没有显着效果。这些结果可以通过生成高度亲核的氧物质来解释，例如与铁螯合物配位的过氧化物，其攻击附近的羰基碳。
Site-specific cleavage of proteins with metal chelates is an approach for designing artificial proteolytic reagents that are directed by proximity to a peptide bond rather than by an amino acid residue type. In the presence of ascorbate and H2O2, an iron chelate attached to Cys-212 of the enzyme human carbonic anhydrase I quickly cleaved the protein between residues Leu-189 and Asp-190 to produce two discrete fragments. The transfer of an 18O atom from [18O]H2O2 (or [18O]O2) to the carboxyl group of Leu-189 was demonstrated by mass spectrometry. Quantitative experiments revealed that one molecule of H2O2 and one molecule of ascorbate afforded the hydrolysis of one peptide bond (1:1:1 stoichiometry) and that the reaction required ascorbate and H2O2. The process is catalytic, since related experiments on the protein bovine serum albumin revealed two cleavage events for each polypeptide chain cleaved. Hydroxyl radical scavengers had no significant effect. These results may be explained by generation of a highly nucleophilic oxygen species, such as peroxide coordinated to the iron chelate, that attacks a carbonyl carbon nearby.