ANALYSIS OF GIARDIA PYROPHOSPHATE DEPENDENT KINASES

贾第鞭毛虫焦磷酸依赖性激酶的分析

• 批准号: 3455686
• 负责人: NELSON F B PHILLIPS
• 金额: $ 9.85万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455686
• 关键词: 6 phosphofructokinase; Giardia; Krebs' cycle; adenosine triphosphate; affinity labeling; aminoacid analyzer; biochemical evolution; bioenergetics; chemical binding; electrofocusing; enzyme mechanism; enzyme structure; enzyme substrate analog; enzyme substrate complex; gel electrophoresis; high energy compound; high performance liquid chromatography; hydrolysis; molecular site; protein purification; protein structure function; pyrophosphates; pyruvate kinase; tissue /cell culture

The protozoan, Giardia lamblia, belongs to the archezoan subkingdom. Some primitive organisms of this subkingdom including Giardia have been found to utilize pyrophosphate (PPi) rather than ATP as an energy source in certain glycolytic reactions. It is hypothesized that these glycolytic enzymes represent 'metabolic fossils' and that PPi may have served as primitive high energy compound. The overall objective of the proposed work is to characterize these PPi dependent enzymes so as to understand the specific molecular structures that result in the preferential utilization of the high energy phosphoryl bond of PPi vs that of ATP. The proposed work will be focused on detail characterization of two such enzymes that have been detected in Giardia, a PPi dependent phosphofructokinase and a pyruvate phosphate dikinase. The specific aims include isolation and characterization of the molecular weight properties, kinetic parameters and regulation by metabolic effectors on these two enzymes. These analyses could further our understanding of energy transfer through phosphoryl bond hydrolysis using the simplest high energy phosphate compound like PPi. Structural characterization of the two enzymes will involve probing the PPi binding and catalytic domains using potential affinity analogs of PPi like azidonitrophenyl pyrophosphate, ferrate ion and pyridoxal phosphate. Chemical modification and steady state kinetics will be used to analyze the binding and catalysis of PPi and ATP. These affinity labels will be used as probes to isolate tryptic peptides at or around the PPi binding and catalytic sites by reverse phase HPLC. The amino acid sequences of these peptides will be determined by automated gas phase sequence analysis. The purpose will be to understand the nature of PPi binding and catalysis in terms of enzyme structure and function. The second objective is to determine if there are similarities in the PPi binding site and the ATP binding sites of higher eukaryotes which will provide evidence for the hypothesis that enzymes utilized PPi as a primitive energy source and that the PPi binding and/or catalytic site evolved to the present form of enzymes that utilize ATP. Finally, the detailed understanding of the PPi- utilizing enzymes that are crucial to Giardia but absent in the host, may be the targets and basis for the rational design of chemotherapeutic drugs against Giardial infections.

原生动物贾迪亚·兰布利亚（Giardia Lamblia）属于Archezoan Subkingdom。 一些 该亚象征的原始生物包括贾第鞭毛虫在内 利用焦磷酸（PPI）而不是ATP作为某些能源 糖酵解反应。 假设这些糖酵解酶 代表“代谢化石”，PPI可能是原始的 高能化合物。 拟议工作的总体目标是 表征这些依赖性PPI的酶以了解特定 导致优先利用的分子结构 PPI与ATP的高能磷酸键。 拟议的工作将 专注于两个已经是这种酶的细节表征 在贾迪亚（Giardia），依赖性磷酸果果酶和丙酮酸中检测到 磷酸二酮酶。 具体目的包括隔离和 分子量特性，动力学参数和 代谢效应子对这两种酶的调节。 这些分析 我们可以进一步理解通过磷酸键的能量转移 使用最简单的高能磷酸化合物（如PPI）的水解。 两种酶的结构表征将涉及探测PPI 使用PPI的潜在亲和力类似物的结合和催化域 偶氮硝基苯基焦磷酸盐，铁酸离子和吡啶毒素磷酸盐。 化学修饰和稳态动力学将用于分析 PPI和ATP的结合和催化。 这些亲和力标签将被使用 作为分离PPI结合或周围的胰蛋白酶肽的探针和 反向相HPLC催化位点。 这些的氨基酸序列 肽将通过自动气相序列分析确定。 这 目的是了解PPI结合和催化的性质 酶结构和功能的术语。 第二个目标是 确定PPI结合位点和ATP中是否有相似之处 较高真核生物的约束地点，将为 假设酶利用PPI作为原始能源，并且 PPI结合和/或催化位点演变为当前形式的 利用ATP的酶。 最后，对PPI的详细理解 利用对贾第鞭毛虫至关重要但在宿主中不存在的酶，可能 成为化学治疗药物合理设计的目标和基础 反对贾第鞭毛虫感染。