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Enzymatic kinetic parameters for polyfluorinated alkyl phosphate hydrolysis by alkaline phosphatase
Author(s) -
Jackson Derek A.,
Mabury Scott A.
Publication year - 2012
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1002/etc.1922
Subject(s) - chemistry , phosphate , hydrolysis , alkyl , alkaline phosphatase , reaction rate constant , alcohol , kinetics , substrate (aquarium) , medicinal chemistry , michaelis–menten kinetics , phosphatase , enzyme kinetics , enzyme , chromatography , nuclear chemistry , organic chemistry , enzyme assay , active site , physics , oceanography , quantum mechanics , geology
The hydrolysis kinetics of three polyfluorinated alkyl phosphate monoesters (monoPAPs), differing in fluorinated chain length, were measured using bovine intestinal alkaline phosphatase to catalyze the reaction. Kinetic values were also measured for analogous hydrogenated phosphate monoesters to elucidate the effects of the fluorinated chain on the rate of enzymatic hydrolysis. Michaelis constants ( K m ) were obtained by a competition kinetics technique in the presence of p ‐nitrophenyl phosphate (PNPP) using UV‐vis spectroscopy. Compared with K m (PNPP), Michaelis constants for monoPAPs ranged from 0.9 to 2.1 compared with hydrogenated phosphates, which ranged from 4.0 to 13.0. Apparent bimolecular rate constants ( k cat / K m ) were determined by monitoring rates of product alcohol formation at low substrate concentrations using gas chromatography–mass spectrometry. The experimental values for k cat / K m averaged as 1.1 × 10 7 M −1 s −1 for monoPAPs compared with 3.8 × 10 5 M −1 s −1 for hexyl phosphate. This suggests that the electron‐withdrawing nature of the fluorinated chain enhanced the alcohol leaving group ability. The results were used in a simple model to suggest that monoPAPs in a typical mammalian digestive tract would hydrolyze in approximately 100 s, supporting a previous study that showed its absence after a dosing study in rats. Environ. Toxicol. Chem. 2012; 31: 1966–1971. © 2012 SETAC