TRANSITION-STATE STRUCTURES FOR ENZYMATIC AND ALKALINE PHOSPHOTRIESTER HYDROLYSIS

The primary and secondary O-18 isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [N-15, phosphoryl-O-18]-, [N-15, phenolic-O-18]-, and [N-15]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary O-18 isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a S(N)2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary O-18 isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The O-18 isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary O-18 isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.