Roos Goedele, Loverix Stefan, Brosens Elke, Van Belle Karolien, Wyns Lode, Geerlings Paul, Messens Joris
Vrije Universiteit Brussel, Algemene Chemie, Pleinlaan 2, 1050, Brussels, Belgium.
Chembiochem. 2006 Jun;7(6):981-9. doi: 10.1002/cbic.200500507.
The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.
pI258 砷酸盐还原酶（ArsC）将砷酸盐还原为亚砷酸盐的过程，是一个亲核取代反应与独特的分子内二硫键级联反应的结合。在这个反应机制中，氧化当量从活性位点转移到 ArsC 的表面。pI258 ArsC 还原砷酸盐的第一个反应步骤是由 Cys10 对二阴离子砷酸盐进行亲核取代反应。第二步涉及 Cys82 对第一步反应中形成的 Cys10 - 砷酸中间体的亲核攻击。本文通过在密度泛函理论背景下使用量子化学计算来研究第二步反应的起始过程。ArsC 催化位点（序列基序：Cys10 - Thr11 - Gly12 - Asn13 - Ser14 - Cys15 - Arg16 - Ser17）中 Cys10 - 砷酸加合物的优化几何结构构成了所有后续计算的起点。热力学数据以及软硬酸碱（HSAB）反应性分析表明，对由 Ser17 稳定的单阴离子 Cys10 - 砷酸加合物存在优先亲核攻击。pI258 ArsC 的 P 环活性位点首先激活一个羟基，随后激活亚砷酸盐作为离去基团，这从这些基团从气相到溶剂相再到酶环境时计算出的亲核离去能力增加可以明显看出。此外，酶环境通过存在由 Cys82 和 Cys89 侧翼的八残基α螺旋（氧化还原螺旋）以及与 Thr11 的氢键，使亲核试剂 Cys82 的硫醇盐形式稳定 3.3 个 pH 单位。与野生型 ArsC 相比，Thr11Ala 突变体的 kcat 值降低，证实了 Thr11 在 Cys82 的 pKa 调节中的重要性。在最后一个反应步骤中，氧化还原螺旋的结构改变将 Cys89 激活为亲核试剂，该氧化还原螺旋作为 Cys89 的 pKa 控制开关；这最后一步对于暴露 Cys82 - Cys89 二硫键是必要的。
