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首页> 外文期刊>Biochemistry >Mechanism-based inhibitors for the inactivation of the bacterial phosphotriesterase
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Mechanism-based inhibitors for the inactivation of the bacterial phosphotriesterase

机译：基于机制的细菌磷酸三酯酶灭活抑制剂

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摘要

1-Bromovinyl (I), Z-2-bromovinyl (II), 1,2-dibromoethyl (III), and a series of 4-(halomethyl)-2-nitrophenyl (IVa-c) diethyl phosphate esters were examined as substrates and mechanism-based inhibitors for the bacterial phosphotriesterase. All of these compounds were found to act as substrates for the enzyme. Inhibitor I rapidly inactivated the enzyme within 1 min, giving a partition ratio of 230. The newly formed covalent adduct with inhibitor I was susceptible to hydrolysis at elevated values of pH and dissociation by NH2OH. Azide was not able to protect the enzyme from inactivation with inhibitor I, implying that the reactive species was not released into solution prior to the inactivation event. The reactive species was proposed to be either an acyl bromide or a ketene intermediate formed by the enzymatic hydrolysis of inhibitor I. Compounds II and III were shown to be relatively poor substrates of phosphotriesterase and they did not induce any significant inactivation of the enzyme. The inhibitor, 4-(bromomethyl)-2-nitrophenyl diethyl phosphate (IVa), was found to irreversibly inactivate the enzyme with a KI = 7.9 mM and kinact = 1. 2 min-1 at pH 9.0. There was no effect on the rate of inactivation upon the addition of the exogenous nucleophiles, azide, and NH2OH. The species responsible for the covalent modification of the enzyme by IVa was most likely a quinone methide formed by the elimination of bromide from the phenolic intermediate. NMR experiments demonstrated that the quinone methide did not accumulate in solution. The chloro (IVb) and fluoro (IVc) analogues did not inactivate the enzyme. These results suggest that the elimination of the halide ion from the phenolic intermediate largely determines the partition ratio for inactivation.
机译：以1-溴乙烯基（I），Z-2-溴乙烯基（II），1,2-二溴乙基（III）和一系列4-（卤甲基）-2-硝基苯基（IVa-c）磷酸二乙酯作为底物和基于机理的细菌磷酸三酯酶抑制剂。发现所有这些化合物均充当酶的底物。抑制剂I在1分钟内迅速使酶失活，分配比为230。与抑制剂I形成的新共价加合物在pH升高时易水解，并被NH2OH分解。叠氮化物不能保护酶免于被抑制剂I灭活，这意味着在灭活事件发生之前，反应性物种并未释放到溶液中。提议的反应性物质是通过抑制剂I的酶促水解形成的酰基溴或乙烯酮中间体。化合物II和III被证明是磷酸三酯酶的相对较差的底物，并且它们不会诱导该酶的任何显着失活。发现抑制剂4-（溴甲基）-2-硝基苯基磷酸二乙酯（IVa）在pH值为9.0时以KI = 7.9 mM和动态= 1. 2 min-1不可逆地灭活该酶。添加外源亲核试剂，叠氮化物和NH2OH对失活速率没有影响。 IVa对酶进行共价修饰的物种很可能是通过从酚类中间体中消除溴化物而形成的醌甲基化物。 NMR实验表明，醌甲基化物没有在溶液中积累。氯（IVb）和氟（IVc）类似物不会使酶失活。这些结果表明，从酚醛中间体中消除卤离子很大程度上决定了灭活的分配比。

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