Zhu Jinge, Knottenbelt Sushilla, Kirk Martin L, Pei Dehua
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Biochemistry. 2006 Oct 10;45(40):12195-203. doi: 10.1021/bi061434v.
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-elimination 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 (13)C-(1)H 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.
S-核糖基高半胱氨酸酶（LuxS）催化S-核糖基高半胱氨酸（SRH）中的硫醚键断裂，生成高半胱氨酸（Hcys）和4,5-二羟基-2,3-戊二酮（DPD），后者是II型细菌自诱导物（AI-2）的前体。提出的催化机制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以及随后的β-消除步骤，所有这些步骤均由活性位点中的二价金属离子（例如Fe(2+)或Co(2+)）和两种通用酸/碱催化。在广泛的pH条件下，对野生型和各种突变形式的LuxS进行了吸收光谱和电子顺磁共振光谱研究。研究表明，金属结合水的pK(a)为10.4。通过用[3-(13)C]半胱氨酸标记的Zn(2+)取代的大肠杆菌LuxS进行的(13)C-(1)H HSQC NMR实验，确定Cys-83的pK(a)值<6。LuxS的活性形式在Cys-83处含有金属结合水和硫醇盐离子，这与催化过程中金属离子（路易斯酸）和Cys-83（通用酸/碱）的拟议作用一致。最后，活性位点中一个不变的Arg-39被证明至少部分负责稳定Cys-83的硫醇盐阴离子。
