Enzymatic Hydrolysis of Organophosphate Esters

这项建议中描述的研究的主要目标是阐明 一种新的磷酸三酯酶的化学反应机理和三维结构 从Spingobium sp.中鉴定。菌株TCM1(SB-PTE)。许多毒物的高毒性 有机磷酸三酯已被开发为许多商业用途的活性成分。 农业和家用杀虫剂，以及作为超强化学战剂。其他，毒性较小的， 有机磷化合物已被广泛用作阻燃剂、增塑剂和 治疗病毒感染的前药。野生型的催化性能和三维结构 SB-PTE型与来自中国的磷酸三酯酶表现出的显著不同 小型假单胞菌(PD-PTE)或迄今已鉴定的任何其他酶。我们建议利用这一点 酶作为设计和创造新型生物催化剂的模板，可用于 有毒有机磷神经毒剂的检测、销毁和解毒 用作农业和家用杀虫剂、增塑剂和化学战剂。这个 该酶的化学机制将通过测定该酶的立体化学过程来阐明。 与手性底物的反应以及通过监测18-氧标记在底物中的去向和 酵素。这些实验将得到重原子同位素效应测量的补充， 底物/活性分布的测定，以及有效的酶抑制剂的鉴定。在……里面 初步实验成功地实现了Sb-PTE的结晶及其测定 用X射线衍射法测定了其三维结构。进一步的结构将在 重点尝试确定该酶活性部位内底物的结合方式。 这些结构将被用作设计和创造新型酶变体的指南 独特的基板配置文件。将构建突变酶的合理和组合文库 以及那些对有毒有机磷的水解具有更高催化熟练程度的变体 将通过独特的筛选和遴选程序进行鉴定。氨基酸的变化 Sb-Pte突变体的序列将与催化性能的增强和 活动站点内结构的更改。

The primary objective for the research described in this proposal is the elucidation of the chemical reaction mechanism and three-dimensional structure of a novel phosphotriesterase recently identified from the bacterium Spingobium sp. strain TCM1 (Sb-PTE). The high toxicity of many organophosphate triesters has been exploited as the active component in many commercial agricultural and household insecticides and as ultra-potent chemical warfare agents. Other, less toxic, organophosphate compounds have been widely utilized as flame retardants, plasticizers, and as prodrugs for viral infections. The catalytic properties and three-dimensional structure of the wild- type Sb-PTE are significantly different from those exhibited by the phosphotriesterase from Pseudomonas diminuta (Pd-PTE) or any other enzyme identified to date. We propose to utilize this enzyme as a template for the design and creation of new biological catalysts that can be exploited for the detection, destruction, and detoxification of toxic organophosphate nerve agents that are currently being used as agricultural and household insecticides, plasticizers, and chemical warfare agents. The chemical mechanism for this enzyme will be elucidated by determining the stereochemical course of the reaction with chiral substrates and by monitoring the fate of 18-oxygen labels in the substrate and enzyme. These experiments will be complemented by measurement of heavy atom isotope effects, determination of the substrate/activity profile, and the identification of potent enzyme inhibitors. In preliminary experiments we have succeeded in the crystallization of Sb-PTE and determination of its three dimensional structure by X-ray diffraction methods. Further structures will be pursued in a focused attempt to determine the mode of substrate binding within the active site of this enzyme. These structures will be utilized as a guide for the design and creation of novel enzyme variants with unique substrate profiles. Rational and combinatorial libraries of mutant enzymes will be constructed and those variants with enhanced catalytic proficiency for the hydrolysis of toxic organophosphates will be identified through unique screening and selection procedures. The changes in the amino acid sequence of the Sb-PTE mutants will be correlated with enhancements in the catalytic properties and alterations in the structure within the active site.