Department of Chemistry , University of Illinois at Urbana-Champaign , 600 South Mathews Avenue , Urbana , Illinois 61801 , United States.
Carl R. Woese Institute for Genomic Biology , University of Illinois at Urbana-Champaign , 1206 West Gregory Drive , Urbana , Illinois 61801 , United States.
J Am Chem Soc. 2018 Dec 26;140(51):17820-17824. doi: 10.1021/jacs.8b06301. Epub 2018 Dec 12.
Phosphite dehydrogenase (PTDH) catalyzes an unusual phosphoryl transfer reaction in which water displaces a hydride leaving group. Despite extensive effort, it remains unclear whether PTDH catalysis proceeds via an associative or dissociative mechanism. Here, primary H and secondary O kinetic isotope effects (KIEs) were determined and used together with computation to characterize the transition state (TS) catalyzed by a thermostable PTDH (17X-PTDH). The large, normal O KIEs suggest an associative mechanism. Various transition state structures were computed within a model of the enzyme active site and H and O KIEs were predicted to evaluate the accuracy of each TS. This analysis suggests that 17X-PTDH catalyzes an associative process with little leaving group displacement and extensive nucleophilic participation. This tight TS is likely a consequence of the extremely poor leaving group requiring significant P-O bond formation to expel the hydride. This finding contrasts with the dissociative TSs in most phosphoryl transfer reactions from phosphate mono- and diesters.
亚磷酸盐脱氢酶（PTDH）催化一个不寻常的磷酸转移反应，其中水取代了一个氢化物离去基团。尽管进行了广泛的研究，但 PTDH 催化反应是通过缔合机制还是解离机制进行仍然不清楚。在这里，确定了初级 H 和次级 O 动力学同位素效应（KIE），并将其与计算结合使用，以描述热稳定 PTDH（17X-PTDH）催化的过渡态（TS）。大的、正常的 O KIE 表明存在缔合机制。在酶活性位点模型内计算了各种过渡态结构，并预测了 H 和 O KIE 以评估每个 TS 的准确性。该分析表明，17X-PTDH 催化一个具有少量离去基团位移和广泛亲核参与的缔合过程。这种紧密的 TS 可能是由于离去基团非常差，需要形成显著的 P-O 键以排出氢化物。这一发现与大多数从磷酸单酯和二酯进行的磷酸转移反应的解离 TS 形成对比。
