MUTATIONAL ANALYSIS OF A PARASITE PURINE SALVAGE ENZYME

寄生虫嘌呤挽救酶的突变分析

• 批准号: 2833990
• 负责人: ANN E EAKIN
• 金额: $ 25.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2833990
• 关键词: Trypanosoma cruzi; X ray crystallography; active sites; aminoacid; calorimetry; catalyst; enzyme activity; hypoxanthine phosphoribosyltransferase; nucleic acid sequence; polymerase chain reaction; protein binding; protein structure function; purine nucleotides; site directed mutagenesis; surface plasmon resonance

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.

该项目的长期目标是了解参与嘌呤碱基回收的代谢酶的结构功能关系。 本研究的主题酶是克氏锥虫（恰加斯病的病原体）的次黄嘌呤磷酸核糖转移酶 (HPRT)。 在人类中，嘌呤核苷酸的合成存在从头途径以及补救途径。 然而，完全缺乏 HPRT 活性会导致 Lesch-Nyhan 综合征，而部分缺乏则可能导致痛风性关节炎。 相比之下，大多数寄生虫无法通过从头途径合成嘌呤，必须依赖挽救途径中的酶（包括 HPRT）来获取细胞代谢所需的嘌呤。 因此，HPRT 已被确定为化疗药物治疗由多种寄生虫引起的人类疾病的潜在靶点。 最近，我们的实验室解析了锥虫 HPRT 的两种高分辨率晶体结构 - 与产品类似物共结晶的酶的 1.4 埃单位分辨率结构（Focia 等人 - a），以及与主要底物磷酸核糖焦磷酸以闭合、过渡态构象捕获的酶的 1.8 埃单位分辨率结构。 (PRPP) 和次黄嘌呤类似物 (Focia, et al. - b)。 这些结构提供了酶催化反应不同阶段 HPRT 的快照，并能够预测特定氨基酸在反应化学中的作用。对于本文提出的研究，克隆基因的位点特异性替换和饱和诱变，加上所得突变酶的动力学和结构研究，将用于测试基于结构的预测。 最近，我们的实验室开发了一种新系统，可以通过细菌中的互补来选择活性重组 HPRT（Canyuk 等人，正在出版）。 该测定将用于通过从饱和诱变产生的突变 HPRT 随机文库中进行选择，对目标氨基酸的功能作用进行快速评估，这些酶具有足够的活性来拯救遗传缺陷细菌。 选择用于阐明 HPRT 催化机制细节的锥虫酶中的目标氨基酸将包括残基，这些残基 1) 形成一个被证明可在活性位点上闭合的柔性环，2) 侧翼位于活性位点底部的保守非脯氨酸顺式肽，以及 3) 直接与结合的底物和/或金属离子相互作用。 将使用稳态和物理结合方法对所选择的酶的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。这项研究的结果将极大地增强我们对这种重要代谢酶的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药物策略的信息，以治疗对人类造成重大负担的疾病。