TRANSITION-STATE STRUCTURES FOR ENZYMATIC AND ALKALINE PHOSPHOTRIESTER HYDROLYSIS
TRANSITION-STATE STRUCTURES FOR ENZYMATIC AND ALKALINE PHOSPHOTRIESTER HYDROLYSIS
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DOI：
10.1021/bi00244a011
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发表时间：
1991-07-30
期刊：
影响因子：
2.9
通讯作者：
CLELAND, WW
中科院分区：
文献类型：
--
作者：
CALDWELL, SR;RAUSHEL, FM;CLELAND, WW
O，O-对硝基苯基磷酸二乙酯(I)和O，O-二乙基O-(4-氨基甲酰基苯基)磷酸(II)两种磷三酯的碱性(KOH)和酶(磷酸三酯酶)水解的初级和次级O-18同位素效应符合缔合机理，在过渡状态下，O，O-二乙基O-(4-氨基甲酰基苯基)磷酸的键级发生了显著的变化。报道了[N-15，磷酰基-O-18]-，[N-15，酚基-O-18]-和[N-15]-O，O-对硝基苯基二乙酯和O，O-二乙基-(4-氨基甲酰基苯基)磷酸的合成。化合物I的初级和次级O-18同位素效应分别为1.0060和1.0063+/-0.0001，而化合物II的初级和次级O-18同位素效应分别为1.027+/-0.002和1.025+/-0.002。这些同位素效应与氢氧化物的限速加成一致，并为没有稳定的磷烷中间体的S(N)2类过渡态提供了证据。对于化合物I的酶解反应，一级和二级O-18同位素效应很小，分别为1.0020和1.0021+/-0.0004，表明酶反应过程中的化学步骤不是限速的。化合物II酶解的O-18同位素效应分别为1.036+/-0.001和1.0181+/-0.0007，与碱解的O-18同位素效应相当，表明该化学步骤是限速的。化合物II的碱性和酶水解的初级O-18同位素效应的相对大小反映了一种更有利于酶反应的过渡状态。
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.