STRUCTURAL ANALYSIS OF THE HGPRT FROM TRYPANOSOMA CRUZI

嘌呤抢救酶--次黄嘌呤鸟嘌呤磷酸核糖基转移酶 (HGPRT)，已被确定为潜在的药物靶点 治疗一些由寄生虫引起的疾病，包括查加斯病 疾病。在此以基于结构的发现方法描述 克氏锥虫HGPRT的有效抑制剂。大多数 目前正在研究的针对寄生虫HGPRT作用的化合物 作为“颠覆性底物”，被酶转化为 阻止后续酶的核苷酸，而不是直接抑制 HGPRT本身的活动。在这项研究中，重点将放在 锥体酶的结构分析及其与酶的相互作用 抑制剂，而不是颠覆性底物。 最有效的酶抑制剂类型通常是对 预测了反应的过渡态。使用重组酶 在细菌中产生的锥体HGPRT的三维结构将是 由X射线结晶学测定。初始结构将在 与产物类似物结合到活性部位的构象提供直接 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 以提供与酶的过渡状态相关的信息。 克隆基因的定点突变将被用来创造突变 酶可能能够结合两种底物，但可能不能 催化作用。这些突变体将被用来使酶与 两种天然底物都结合在一起。一项免费的战略将采用 不可挽救的嘌呤类似物(其中活性氮被取代 含碳原子)与锥虫共结晶实验 酶和第二底物，磷酸核糖基植磷酸盐。也是 将进行重组酶的共结晶实验 使用当前可用的过渡态类似物。除 结构研究，酶催化反应的机理将 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‘S，底物结合 抑制剂的顺序和表观KI。 这里提出的结构和动力学实验是为 锥虫HGPRT将为设计HGPRT提供有价值的信息 专门针对这种酶的有效抑制剂。此外， 阐明底物结合和催化过程中的结构变化 协助针对其他寄生虫HGPRT的抑制剂设计工作 并将提供新的信息，以增加我们对 酶催化与结构/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.