Significant research effort has been invested into elucidating the mechanism and reactivity of phosphate mono- and diesters, and how their reactions are efficiently catalysed by enzymes. Although both reactions involve the transfer of a phosphoryl group with similar geometries, it is uncommon to find enzymes capable of catalyzing both reactions and the factors governing the selectivity of this enzyme remain poorly understood. Herein, we examine the reactivity of a series of aryl fluorophosphates to study the effect of changing the nature of the transition state while retaining the size and hydrogen bond donor/acceptor properties of the phosphoryl group in the corresponding monoester. We have performed an extensive kinetic and theoretical analysis of the hydrolysis of the reference reaction in solution, demonstrating that fluorophosphates mimic the behaviour of the corresponding methyl aryl phosphate diesters, proceeding through concerted transition states with structures that are sensitive to the acidity of the leaving group. We have also performed an initial study of the catalysis of this reaction by the R166A mutant of alkaline phosphatase, and find that the enzyme has a greater proficiency with the diester substrate than fluorophosphate substrates, although both are greatly reduced relative to the corresponding monoester.
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