Catalytic mechanism of S-ribosylhomocysteinase (LuxS): stereochemical course and kinetic isotope effect of proton transfer reactions.
Catalytic mechanism of S-ribosylhomocysteinase (LuxS): stereochemical course and kinetic isotope effect of proton transfer reactions.
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DOI：
10.1021/bi0491088
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发表时间：
2004-07
期刊：
影响因子：
2.9
通讯作者：
Jinge G. Zhu;Reena Patel;D. Pei
中科院分区：
文献类型：
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作者：
Jinge G. Zhu;Reena Patel;D. Pei
S-核糖基高半胱氨酸酶（LuxS）催化S-核糖基高半胱氨酸（SRH）中的硫醚键裂解以产生高半胱氨酸和4，5-二羟基-2，3-戊二酮（DPD），DPD是II型细菌群体感应分子的前体。所提出的机制涉及一系列的质子转移反应，这是由一个Fe 2+离子和两个一般的酸/碱在LuxS活性位点催化，导致在核糖羰基从其C1到C3的位置迁移。随后在C4和C5位置的β-消除完成催化循环。本工作以氘标记的SRH为底物，通过对反应产物的核磁共振氢谱和质谱分析，确定了质子转移反应的区域化学和立体化学过程。我们的数据表明，在催化过程中，核糖C2质子到其C1位置和C3质子到C2位置的表面转移，而核糖C4质子被完全洗涤到溶剂中。主要的氘动力学同位素效应表明2-酮中间体向3-酮中间体的转化是部分速率限制的。然而，Glu-57（催化中推定的第二通用酸/碱）突变为天冬氨酸使得最终的β-消除步骤速率受限。
S-ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum sensing molecule. The proposed mechanism involves a series of proton-transfer reactions, which are catalyzed by an Fe2+ ion and two general acids/bases in the LuxS active site, resulting in the migration of the ribose carbonyl group from its C1 to C3 position. Subsequent beta-elimination at C4 and C5 positions completes the catalytic cycle. In this work, the regiochemistry and stereochemical course of the proton transfer reactions were determined by carrying out the reactions using various specifically deuterium-labeled SRH as substrate and analyzing the reaction products by 1H NMR spectroscopy and mass spectrometry. Our data indicate a suprafacial transfer of the ribose C2 proton to its C1 position and the C3 proton to the C2 position during catalysis, whereas the ribose C4 proton is completely washed into solvent. The primary deuterium kinetic isotope effect suggests that the conversion of 2-keto intermediate to 3-keto intermediate is partially rate limiting. However, mutation of Glu-57, the putative second general acid/base in catalysis, to an aspartic acid renders the final beta-elimination step rate limiting.