TY - JOUR
T1 - 18 O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer
AU - Howe, Graeme W.
AU - Van Der Donk, Wilfred A.
N1 - Publisher Copyright:
© 2018 American Chemical Society.
PY - 2018/12/26
Y1 - 2018/12/26
N2 - 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.
AB - 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.
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U2 - 10.1021/jacs.8b06301
DO - 10.1021/jacs.8b06301
M3 - Article
C2 - 30525552
AN - SCOPUS:85059540785
SN - 0002-7863
VL - 140
SP - 17820
EP - 17824
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 51
ER -