STRUCTURAL ANALYSIS OF THE HGPRT FROM TRYPANOSOMA CRUZI

一种嘌呤补救酶，次黄嘌呤鸟嘌呤磷酸核糖基转移酶 （HGPRT），已被确定为药物的潜在靶点， 治疗一些寄生虫引起的疾病，包括南美锥虫病 疾病 在本文中描述的基于结构的方法来发现 来自克氏锥虫的HGPRT强效抑制剂。 大部分 目前正在研究的针对寄生虫HGPRT的化合物 作为“颠覆性底物”，被酶转化为修饰的 阻断后续酶的核苷酸，而不是直接抑制 HGPRT本身的活动。 对于这项研究，重点将放在 锥虫酶的结构分析及其与 抑制剂，而不是颠覆性底物。 最有效的酶抑制剂类型通常是酶的模拟物， 预测反应的过渡态。 使用重组酶 在细菌中产生，锥虫HGPRT的三维结构将是 由X射线晶体学确定。 最初的结构将在 产物类似物与活性位点结合的构象提供了直接的 与目前可用的人类酶结构的比较 与产品绑定，GMP。 随后将采用几种方法 以提供与酶的过渡态相关的信息。 克隆基因的定点突变将用于产生突变体 酶可能能够结合两种底物，但可能不能 催化作用 这些突变体将用于使酶与 两种天然基质结合。 一个免费的战略将采用一个 不可挽救的嘌呤类似物（其中活性氮被取代 在与锥虫的共结晶实验中， 酶和第二底物磷酸核糖基磷酸。 也 用重组酶进行共结晶实验 使用当前可用的过渡态类似物。 除了有 结构研究，酶催化反应的机理将 使用稳态动力学进行研究。 这些研究将提供 关于相对反应速率的细节，Km，底物结合 顺序和表观Ki的抑制剂。 本文提出的结构和动力学实验， 锥虫HGPRT将为设计 针对这种酶的强效抑制剂。 此外该 在底物结合和催化过程中结构变化的阐明将 协助针对其他寄生虫的HGPRT的抑制剂设计工作 并将提供新的信息，以增加我们的一般知识， 酶催化和结构/功能关系。

A purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HGPRT), has been identified as a potential targets of drugs for the treatment of a number f diseases cause by parasites, including Chagas' disease. Described herein in a structure-based approach to the discovery of potent inhibitors of HGPRT from Trypanosoma cruzi. Most of the compounds currently being studied that target the HGPRT's of parasites act as "subversive substrates" that are converted by the enzyme into modified nucleotides that block subsequent enzymes, rather than directly inhibiting the activity of the HGPRT itself. For this study, the focus will be on the structural analysis of the trypanosomal enzyme and interactions with inhibitors, rather than subversive substrates. The most potent types of inhibitors of enzymes often are mimics of the predicted transition state of the reaction. Using recombinant enzyme produced in bacteria, the 3-D structure of the trypanosomal HGPRT will be determined by X-ray crystallography. The initial structure will be in conformation with a product analog bound to the active site provide direct comparisons with the currently available structure of the human enzyme bound to product, GMP. Several approaches will subsequently be employed to provide information relevant to the transition state of the enzyme. Site-directed mutagenesis of the cloned gene will be used to create mutant enzymes that may be able to bind both substrates but may be incapable of catalysis. These mutants will be used to co-crystallize the enzyme with both natural substrates bound. A complimentary strategy will employ a nonsalvageable purine analog (in which the reactive nitrogen is replaced with a carbon atom) in co-crystallization experiments with the trypanosomal enzyme and the second substrate, phosphoribosylphyrophospate. Also cocrystallization experiments with the recombinant enzyme will be performed using currently available transition state analogs. In addition to the structural studies, the mechanism for the enzyme catalyzed reaction will be investigate using steady state kinetics. These studies will provide details with regard to relative reaction rates., Km 's, substrate binding order and apparent K i's for inhibitors. The structural and kinetic experiments proposed herein for the trypansosomal HGPRT will contribute valuable information for the design of potent inhibitors specifically targeted to this enzyme. In addition, the elucidation of structural changes during substrate bind and catalysis will assist inhibitor design efforts targeting the HGPRT's of other parasites and will provide new information to increase our general knowledge about enzyme catalysis and structure/function relationships.