The substrate-assisted general base catalysis model for phosphate monoester hydrolysis: Evaluation using reactivity comparisons

Reactions of phosphate monoesters are ubiquitous in biological chemistry. Hence, this class of reactions has been subjected to extensive mechanistic analysis by physical organic chemists seeking to understand the nonenzymatic reactions and to apply this understanding to the corresponding enzymatic reactions. Substrate-assisted general base catalysis of phosphoryl transfer, in which a proton from the nucleophile is transferred to a nonbridging phosphoryl oxygen of the substrate prior to attack, has recently been proposed as a mechanism for both nonenzymatic and enzymatic reactions of phosphate monoester dianions and related compounds, in opposition to the previously accepted mechanism of direct nucleophilic reaction. We have evaluated this new mechanism for the hydrolysis of a phosphate monoester dianion in solution by considering the reactivity of the monoester monoanion that is a reaction intermediate in the proposed proton transfer. The monoanion of the monoester 2,4-dinitrophenyl phosphate (DNPP-) and its diester analogue, methyl 2,4-dinitrophenyl phosphate monoanion (MDNPP-), have similar rate constants for reaction with several nucleophiles (k(rel) = k(DNPP)/k(MDNPP) approximate to 10). In contrast, the substrate-assisted catalysis proposal requires that the rate constant for reaction of hydroxide ion with DNPP- be similar to 10(9)-fold larger than the experimentally determined rate constant for the corresponding reaction of hydroxide ion with MDNPP-. These and additional observations render substrate-assisted general base catalysis an unlikely alternative to the classical mechanism for nonenzymatic phosphoryl transfer.