18 O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer

Graeme W. Howe, Wilfred A. Van Der Donk

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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 2 H and secondary 18 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 18 O KIEs suggest an associative mechanism. Various transition state structures were computed within a model of the enzyme active site and 2 H and 18 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.

Original languageEnglish (US)
Pages (from-to)17820-17824
Number of pages5
JournalJournal of the American Chemical Society
Issue number51
StatePublished - Dec 26 2018


ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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