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

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 isotop...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2018-12, Vol.140 (51), p.17820-17824
Main Authors: Howe, Graeme W, van der Donk, Wilfred A
Format: Article
Language:English
Subjects:
Catalysis
Catalytic Domain
Deuterium - chemistry
Kinetics
Models, Chemical
Models, Molecular
NADH, NADPH Oxidoreductases - chemistry
Oxygen Isotopes - chemistry
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Summary: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.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.8b06301