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¹⁸O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer
- Howe, Graeme W., van der Donk, Wilfred A.
- Journal of the American Chemical Society 2018 v.140 no.51 pp. 17820-17824
- Lewis bases, active sites, catalytic activity, chemical bonding, deuterium, enzymes, hydrides, models, oxygen, phosphates, stable isotopes, thermal stability
- 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 ²H and secondary ¹⁸O 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 ¹⁸O KIEs suggest an associative mechanism. Various transition state structures were computed within a model of the enzyme active site and ²H and ¹⁸O 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.