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首页> 外文期刊>Biochemistry >Catalytic Mechanism of S-Ribosylhomocysteinase: Ionization State of Active-Site Residues
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Catalytic Mechanism of S-Ribosylhomocysteinase: Ionization State of Active-Site Residues

机译：S-核糖基同型半胱氨酸酶的催化​​机理：活性位点残基的电离状态

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摘要

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial autoinducer (AI-2). The proposed catalytic mechanism involves two consecutive ribose carbonyl migration steps via an intramolecular redox reaction and a subsequent beta-elimi-nation step, all catalyzed by a divalent metal ion (e.g., Fe~(2+) or Co~(2+)) and two general acids/bases in the active site. Absorption and EPR spectroscopic studies were performed with both wild-type and various mutant forms of LuxS under a wide range of pH conditions. The studies revealed a pK_a of 10.4 for the metal-bound water. The pK_a value of Cys-83 was determined to be <6 by ~(1)3C-~1H HSQC NMR experiments with [3-~(13)C]cysteine-labeled Zn~(2+)-substituted Escherichia coli LuxS. The active form of LuxS contains a metal-bound water and a thiolate ion at Cys-83, consistent with the proposed roles of the metal ion (Lewis acid) and Cys-83 (general acid/base) during catalysis. Finally, an invariant Arg-39 in the active site was demonstrated to be at least partially responsible for stabilizing the thiolate anion of Cys-83.
机译：S-核糖基同型半胱氨酸酶（LuxS）催化S-核糖基同型半胱氨酸（SRH）中硫醚键的裂解，产生高半胱氨酸（Hcys）和4,5-二羟基-2,3-戊二酮（DPD），II型细菌自诱导物的前体AI-2）。所提出的催化机理涉及通过分子内氧化还原反应的两个连续的核糖羰基迁移步骤和随后的β-消除步骤，所有这些步骤均由二价金属离子（例如Fe〜（2+）或Co〜（2+））催化以及活性部位中的两种通用酸/碱。野生型和各种突变形式的LuxS在广泛的pH条件下均进行了吸收和EPR光谱研究。研究表明与金属结合的水的pK_a为10.4。通过[（〜（13）C]半胱氨酸标记的Zn〜（2+）取代的大肠杆菌LuxS的〜（1）3C ~~ 1H HSQC NMR实验，确定Cys-83的pK_a值<6。 LuxS的活性形式在Cys-83处包含与金属结合的水和硫醇根离子，这与金属离子（路易斯酸）和Cys-83（通用酸/碱）在催化过程中的拟议作用一致。最后，活性位点的不变Arg-39被证明至少部分负责稳定Cys-83的硫醇根阴离子。

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