Caldwell S R, Raushel F M, Weiss P M, Cleland W W
Department of Chemistry, Texas A&M University, College Station 77843.
Biochemistry. 1991 Jul 30;30(30):7444-50. doi: 10.1021/bi00244a011.
The primary and secondary 18O 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 [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O 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 SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O 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 18O 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 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.
两种磷酸三酯，即O,O - 二乙基对硝基苯基磷酸酯（I）和O,O - 二乙基O -（4 - 氨基甲酰基苯基）磷酸酯（II），在碱性（KOH）和酶促（磷酸三酯酶）水解过程中的一级和二级18O同位素效应，与一种缔合机制一致，该机制在过渡态中磷酰基和酚类离去基团氧的键级发生了显著变化。描述了[15N,磷酰基 - 18O] -、[15N,酚类 - 18O] - 和[15N] - O,O - 二乙基对硝基苯基磷酸酯以及O,O - 二乙基O -（4 - 氨基甲酰基苯基）磷酸酯的合成。化合物I碱性水解的一级和二级18O同位素效应分别为1.0060和1.0063±0.0001，而化合物II的分别为1.027±0.002和1.025±0.002。这些同位素效应与氢氧根的限速加成一致，并为不存在稳定的磷叶立德中间体的类似SN2过渡态提供了证据。对于化合物I的酶促水解，一级和二级18O同位素效应非常小，分别为1.0020和1.0021±0.0004，这表明酶促机制中的化学步骤不是限速步骤。化合物II酶促水解的18O同位素效应分别为1.036±0.001和1.0181±0.0007，其大小与碱性水解的同位素效应相当，表明化学步骤是限速步骤。化合物II碱性和酶促水解的一级18O同位素效应的相对大小反映了酶促反应过渡态的进展程度更高。
