MUTATIONAL ANALYSIS OF A PARASITE PURINE SALVAGE ENZYME

这个项目的长期目标是了解参与挽救嘌呤碱基的代谢酶的结构-功能关系。这项研究的主题是查加斯病的病原体克氏锥虫的次黄嘌呤磷酸核糖基转移酶(HPRT)。在人类中，存在合成嘌呤核苷酸的从头合成途径和挽救途径。然而，完全缺乏HPRT活性是Lesch-Nyhan综合征的原因，而部分缺乏HPRT会导致痛风性关节炎。相反，大多数寄生虫不能通过从头途径合成嘌呤，必须依赖挽救途径中的酶，包括HPRT，以获得细胞新陈代谢所需的嘌呤。因此，HPRT已被确定为几种寄生虫引起的人类疾病的化疗药物的潜在靶点。最近，我们实验室解决了锥虫HPRT的两个高分辨晶体结构--1.4埃单位的酶的分辨结构与产物类似物共结晶(Focia等人。-a)和在与初级底物磷酸核糖焦磷酸(PRPP)和次黄嘌呤类似物(Focia等人)封闭的过渡态构象中捕获的酶的1.8埃单位的分辨结构(Focia等人。-b)。这些结构提供了酶催化反应不同阶段的HPRT的快照，并能够预测特定氨基酸在反应化学中的作用。在这里介绍的研究中，克隆基因的定点替换和饱和突变，以及对所产生的突变酶的动力学和结构研究，将被用来检验基于结构的预测。最近，我们实验室开发了一种新的系统，它能够通过在细菌中互补来选择具有活性的重组HPRT(Canyuk等人，在出版社中)。这项检测将被用来快速评估目标氨基酸的功能作用(S)，方法是从饱和突变产生的突变HPRT的随机文库中选择那些具有足够活性的酶来拯救遗传缺陷细菌。用来阐明HPRTS催化机制细节的锥体酶中的目标氨基酸将包括：1)形成一个靠近活性部位的柔性环，2)活性部位底面上保守的非脯氨酸顺式多肽，3)直接与结合底物和/或金属离子相互作用。将使用稳态和物理结合方法对选定的酶的突变形式进行动力学表征，并在适当的情况下确定突变酶的晶体结构。这项研究的结果将极大地加深我们对这种重要代谢酶结构与功能关系的理解。对公众的好处包括更好地了解人类疾病的分子基础，以及提供可用于设计治疗对人类造成重大负担的疾病的药物的战略的信息。

The long range goal of this project is to understand structure- function relationships for a metabolic enzyme involved in the salvage of purine bases. The enzyme that is the subject of this study is the hypoxanthine phosphoribosyltransferase (HPRT) of Trypanosoma cruzi, etiologic agent of Chagas' disease. In humans, de novo, as well as salvage pathways exist for the synthesis of purine nucleotides. However, the complete absence HPRT activity is responsible for Lesch-Nyhan syndrome, while a partial deficiency can result in gouty arthritis. In contrast, most parasites are unable to synthesize purines via de novo pathways, and must rely on enzymes in salvage pathways, including HPRTs, for the purines needed in cellular metabolism. Thus, HPRTs have been identified as potential targets for drugs in the chemotherapeutic treatment of human disease caused by several species of parasites. Recently, two high resolution crystal structures of the trypanosomal HPRT were solved in our laboratory - a 1.4 Angstrom units resolution structure of the enzyme co-crystallized with a product analog (Focia, et al. - a) and a 1.8 Angstrom units resolution structure of the enzyme captured in a closed, pre-transition state conformation with the primary substrate, phosphoribosylpyrophosphate (PRPP) and a hypoxanthine analog (Focia, et al. - b). These structures provide snapshots of an HPRT at different stages of the enzyme-catalyzed reaction and enable predictions for the roles of specific amino acids in the chemistry of the reaction. For the studies presented herein, site-specific replacement and saturation mutagenesis of the cloned gene, coupled with kinetic and structural studies of the resultant mutant enzymes, will be used to test the structure-based predictions. Recently, a novel system was developed in our laboratory that enables the selection for active recombinant HPRTs by complementation in bacteria (Canyuk et al., in press). This assay will be used to provide a rapid assessment of the functional role(s) for target amino acids by selecting from random libraries of mutant HPRTs created by saturation mutagenesis, those enzymes with sufficient activity to rescue the genetically deficient bacteria. Target amino acids in the trypanosomal enzyme chosen to illuminate details of the catalytic mechanism of HPRTs will include residues that 1) form a flexible loop demonstrated to close over the active site, 2) flank a conserved non-proline cis-peptide on the floor of the active site, and 3) interact directly with bound substrates and/or metal ions. Selected mutant forms of the enzyme will be characterized kinetically, using steady-state and physical binding methods, and where appropriate, crystal structures of the mutant enzymes will be determined. The results of this study will greatly enhance our understanding of structure-function relationships for this important metabolic enzyme. Benefits for the public include a better understanding of the molecular basis of human disease as well as the provision of information that could be used in strategies for the design of drugs to treat diseases which are a significant burden to human kind.