18O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer.
18O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer.
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18O 动力学同位素效应揭示了亚磷酸脱氢酶催化磷酰基转移的缔合过渡态。
DOI：
10.1021/jacs.8b06301
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发表时间：
2018
影响因子：
15
通讯作者：
vanderDonk,WilfredA
中科院分区：
文献类型：
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作者：
Howe,GraemeW;vanderDonk,WilfredA
亚磷酸脱氢酶（PTDH）催化一种不寻常的磷酰基转移反应，其中水取代氢化物离去基团。尽管广泛的努力，它仍然不清楚PTDH催化是否通过缔合或解离机制进行。在这里，初级2 H和次级18 O动力学同位素效应（KIE）的测定和计算一起使用，以表征由热稳定的PTDH（17 X-PTDH）催化的过渡态（TS）。大的、正常的18 O KIE表明存在一种联想机制。在酶活性中心模型内计算了各种过渡态结构，并预测了2 H和18 O的KIE，以评估每个TS的准确性。该分析表明，17 X-PTDH催化缔合过程，具有很少的离去基团置换和广泛的亲核参与。这种紧密的TS可能是极差的离去基团需要显著的P-O键形成以排出氢化物的结果。这一发现与大多数磷酸单酯和二酯的磷酰基转移反应中的解离TS形成对比。
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, primary2H and secondary18O 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, normal18O KIEs suggest an associative mechanism. Various transition state structures were computed within a model of the enzyme active site and2H and18O 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.