18O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer.
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ABSTRACT: 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.
SUBMITTER: Howe GW
PROVIDER: S-EPMC6467793 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
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