Mechanistic and Stereochemical Studies of Glycine Oxidase from Bacillus subtilis Strain R5
Mechanistic and Stereochemical Studies of Glycine Oxidase from Bacillus subtilis Strain R5
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DOI：
10.1021/bi100553n
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发表时间：
2010-08-31
期刊：
影响因子：
2.9
通讯作者：
Akhtar, Muhammad
中科院分区：
文献类型：
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作者：
Jamil, Farrukh;Gardner, Qurra-tul-Ann Afza;Akhtar, Muhammad
从枯草芽孢杆菌中克隆甘氨酸氧化酶基因，并在大肠杆菌中表达。通过质谱法发现纯化的酶具有40763的蛋白质：11（从DNA序列预测的值为40761.6）和785.1的FAD辅基M-r（理论值为785.5）。甘氨酸氧化酶最佳地催化甘氨酸和氧转化为乙醛酸、过氧化氢和氨。使用[2-RS-H-3（2），2-C-14]-、[2-R-H-3，2-C-14]-和[2-S-H-3，2-C-14]甘氨酸样品，我们发现在整个过程中H-si被除去。在厌氧条件下将酶与[2-RS-H-3（2），2-C-14]甘氨酸一起孵育，当仅发生还原性一半反应时，导致98.5%的原始甘氨酸的回收，其具有与起始底物相同的H-3：C-14比率。用[2-H-2（2）]甘氨酸研究了初级同位素效应，发现对质子和氘底物的专一性常数k（cat）/K-M分别为1.46 × 10（3）和1.05 × 10（2）M-1 s（-1）。已经考虑了含FAD氧化酶的两种替代机制，其涉及FADH（2）-亚氨基酸复合物的中间体或通过碳负离子形成的共价连接至FAD的氨基酸。目前的知识的机制进行了审查，我们认为，涉及FADH（2）-亚氨基酸复合物的机制可以被解剖，令人满意地解释一些令人困惑的观察，碳负离子机制最初设想。此外，我们的研究结果，连同在文献中的观察，表明甘氨酸与酶的相互作用发生在一个紧密的三元复合物，这是保护从介质的质子。
Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein :11, of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M-r of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-H-3(2),2-C-14]-, [2-R-H-3.2-C-14]- and [2-S-H-3,2-C-14]glycine, we found that in the overall process H-si is removed. Incubation of the enzyme with [2-RS-H-3(2),2-C-14]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same H-3:C-14 ratio as the starting substrate. The primary isotope effect was studied using [2-H-2(2)]glycine, and we found that the specificity constants, k(cat)/K-M, for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M-1 s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.