Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase
Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase
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DOI：
10.1021/bi0497805
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发表时间：
2004-05-18
期刊：
影响因子：
2.9
通讯作者：
Raushel, FM
中科院分区：
文献类型：
--
作者：
Aubert, SD;Li, YC;Raushel, FM
微小假单胞菌的磷酸三酯酶(PTE)是一种锌金属酶，能降解多种有机磷化合物。测定了锌/锌、镉/镉、混合金属锌/镉杂化PTE在不同底物上的动力学参数，确定了各金属离子的结合和催化作用。对对硝基苯基磷酸二乙酯(I)和对氯苯基磷酸二乙酯(II)的pH值曲线表明，在pH为5-10的范围内，单一基团的电离对底物的周转是关键的。由动力学分析确定的pK(A)值取决于占据双核金属中心内的α位置的金属离子的同一性。这些结果表明，通过与(x-金属离子)相连的水分子的电离，水解性亲核试剂以氢氧化物的形式被激活。测定了对氯苯硫代磷酸二乙酯(IV)和对氯苯硫代磷酸二乙酯(IV)的金属取代形式的动力学常数。IV的动力学常数大于II的动力学常数。反向硫代效应与磷酰氧/硫键通过直接连接到金属中心的极化是一致的。当Cd~(2+)占据β-金属离子位置时，速率增强作用更大。用一系列丙氨酸和天冬酰胺突变来表征Asp233、His254和Asp301的催化作用。Asp233或His254的突变导致反应迟缓的底物对氯苯基磷酸二乙酯的水解率增加，对氧磷的动力学常数降低。这些结果与从Asp301到His254再到Asp233的质子继电器的存在一致，该质子继电器用于将质子从活性中心转移到不需要离开基团苯酚的底物上。提出了细菌PTE降解有机磷的机理。
Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the (x-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd2+ occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.