Rapid and Quantitative Disulfide Bond Formation for a Polypeptide Chain Using a Cyclic Selenoxide Reagent in an Aqueous Medium
Rapid and Quantitative Disulfide Bond Formation for a Polypeptide Chain Using a Cyclic Selenoxide Reagent in an Aqueous Medium
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DOI：
10.1002/chem.201002742
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发表时间：
2011-01-01
影响因子：
4.3
通讯作者：
Iwaoka, Michio
中科院分区：
文献类型：
--
作者：
Arai, Kenta;Dedachi, Kenichi;Iwaoka, Michio
为了阐明多肽分子与水溶性硒氧化物试剂反式-3，4-二羟基硒杂环戊烷氧化物（DHSox）的二硫键（SS）形成反应的反应机理，在pH 7.0和25 ℃下对重组水蛭素CX-397变体的还原态（R）进行短期氧化实验。在反应中，R依次氧化为一个SS，两个SS，和三个SS中间合奏1分钟内。动力学分析表明，SS形成的三个二阶速率常数是成比例的反应物SS中间体中存在的硫醇基团的数量，表明SS形成的随机性。在HF/6-31 +G（d，p）水平上，采用极化连续模型进行从头计算，结果表明SS的生成反应是高度放热的，并通过一个具有扭曲的线性O-Se-S键的硫硒铀中间体进行.结果清楚地表明，SS形成反应的速率决定步骤是硫醇底物和DHSox之间的第一个双分子过程，而不是随后释放SS产物的过程。
To elucidate the reaction mechanism of the disulfide (SS) bond formation reaction of a polypeptide molecule with a water-soluble selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), short-term oxidation experiments were carried out for the reduced state (R) of a recombinant hirudin CX-397 variant at pH 7.0 and 25 degrees C. In the reaction, R was oxidized sequentially to one-SS, two-SS, and three-SS intermediate ensembles within 1 min. The kinetic analysis revealed that the three second-order rate constants for the SS formation are proportional to the number of thiol groups existing in the reactant SS intermediates, indicating the stochastic nature of the SS formation. Ab initio calculation at the HF/6-31 +G(d,p) level in water by using the polarizable continuum model suggested that the SS formation reaction is highly exothermic and proceeds via a reactive thioselenurane intermediate with a distorted linear O-Se-S linkage. The results clearly demonstrated that the rate-determining step of the SS formation reaction is the first bimolecular process between a thiol substrate and DHSox rather than the subsequent process to release a SS product.