MUTATIONAL ANALYSIS OF A PARASITE PURINE SALVAGE ENZYME

这个项目的长期目标是了解嘌呤碱基抢救中所涉及的代谢酶的结构-功能关系。 作为本研究的主题的酶是次黄嘌呤磷酸核糖基转移酶（HPRT）的锥虫克氏锥虫，南美锥虫病的病原体。 在人类中，嘌呤核苷酸的合成存在从头途径和补救途径。 然而，HPRT活性的完全缺失是Lesch-Nyhan综合征的原因，而部分缺乏则可导致痛风性关节炎。 相反，大多数寄生虫不能通过从头途径合成嘌呤，并且必须依赖于补救途径中的酶，包括HPRT，用于细胞代谢所需的嘌呤。 因此，HPRT已被确定为药物在由几种寄生虫引起的人类疾病的化学治疗中的潜在靶标。 最近，在我们的实验室中解决了锥虫HPRT的两个高分辨率晶体结构-与产物类似物共结晶的酶的1.4埃单位分辨率结构（Focia，et al. - a）和以封闭的前过渡态构象捕获的酶的1.8埃单位分辨率结构，磷酸核糖焦磷酸（PRPP）和次黄嘌呤类似物（Focia，et al. - B）。 这些结构提供了HPRT在酶催化反应的不同阶段的快照，并能够预测特定氨基酸在反应化学中的作用。对于本文所述的研究，将使用克隆基因的位点特异性置换和饱和诱变以及所得突变酶的动力学和结构研究来测试基于结构的预测。 最近，在我们的实验室中开发了一种新的系统，该系统能够通过细菌中的互补作用来选择活性重组HPRT（Rumuk等人，出版中）。 该试验将用于通过从饱和诱变产生的突变HPRT随机文库中选择具有足够活性以拯救遗传缺陷细菌的酶，快速评估靶氨基酸的功能作用。 选择用于阐明HPRT催化机制细节的锥虫酶中的靶氨基酸将包括以下残基：1）形成柔性环，该柔性环被证明在活性位点上闭合，2）在活性位点底部的保守非脯氨酸顺式肽的侧翼，以及3）与结合的底物和/或金属离子直接相互作用。 将使用稳态和物理结合方法对酶的选定突变形式进行动力学表征，并且在适当的情况下，将确定突变酶的晶体结构。这项研究的结果将大大提高我们对这种重要的代谢酶的结构-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药物治疗对人类造成重大负担的疾病的战略的信息。

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.