Expression and Structure of Soluble Human Nucleotidases

可溶性人核苷酸酶的表达和结构

• 批准号: 7106799
• 负责人: TERENCE LEE KIRLEY
• 金额: $ 3.26万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-14 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106799
• 关键词: X ray crystallography; acid anhydride hydrolase; active sites; affinity labeling; computer simulation; conformation; disulfide bond; enzyme activity; enzyme mechanism; enzyme structure; hydrolysis; nucleotidases; protein engineering; protein folding; protein purification; pyrophosphatase; site directed mutagenesis

DESCRIPTION (provided by applicant): Human nucleotidases are enzymes that terminate purinergic receptor-mediated responses, including many processes dependent upon extracellular ATP and ADP. The potential clinical utility of injecting soluble nucleotidases therapeutically to modulate these physiologically important processes has already been demonstrated. One specific therapeutic use of such soluble nucleotidases is the control of the pathophysiological blood clotting that causes heart attacks and strokes, via hydrolysis of ADP that triggers platelet activation and subsequent blood coagulation. Thus, one goal of the proposed research is to elucidate the structures of the soluble human nucleotidases and to use this information to understand their functions. A second goal is to use the structural information to design modified nucleotidases, having increased activity and specificity for nucleotides modulating physiological processes. Elucidating the structural determinants of substrate hydrolysis and specificity is crucial for control of important, purinergically controlled, processes. The two known, naturally occurring, soluble forms of human ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) will be expressed in bacteria and refolded to generate large quantities of enzymatically active, soluble eNTPDases. The expressed proteins will be purified, enzymatically characterized, and crystallized to determine their structures. Affinity labeling, site-directed mutagenesis, and computer modeling will be used to determine the roles played by individual amino acid residues in nucleotide hydrolysis, as well as to identify residues important for nucleotidase specificity. These results will be compared and contrasted with similar results obtained using another soluble human nucleotidase, newly discovered by our laboratory, which is related in sequence to a nucleotidase found in the saliva of bloodsucking insects, but unrelated in sequence to the eNTPDases. The study of this nucleotidase is important for two reasons. First, it represents a second potential therapeutic avenue for developing therapeutic soluble nucleotidases. Second, it will demonstrate how two different protein primary structures are used to do the same enzymatic task, aiding in the design of more efficacious therapeutic soluble nucleotidases.

描述（由申请人提供）：人核苷酸酶是终止嘌呤能受体介导的反应的酶，包括许多取决于细胞外ATP和ADP的过程。已经证明了对这些重要重要过程调节这些重要过程的可溶性核苷酸酶的潜在临床实用性。这种可溶性核苷酸酶的一种特定治疗用途是通过水解ADP的水解来控制导致心脏病发作和中风的病理生理血液，从而触发血小板激活以及随后的血液凝结。因此，拟议的研究的一个目标是阐明可溶性人核苷酸酶的结构，并使用这些信息来理解其功能。第二个目标是使用结构信息来设计改良的核苷酸酶，对调节生理过程的核苷酸的活性和特异性增加。阐明底物水解和特异性的结构决定因素对于控制重要的，嘌呤能控制的过程至关重要。 人类外生核苷三磷酸二磷酸氢集酶（ENTPDase）的两种已知，天然存在的可溶性形式将在细菌中表达，并重新折叠以产生大量酶活性活性，可溶性entpdase。表达的蛋白质将被纯化，酶促表征并结晶以确定其结构。亲和力标记，定位诱变和计算机建模将用于确定单个氨基酸残基在核苷酸水解中起起的作用，并确定对核苷酸酶特异性重要的残基。这些结果将与使用另一个可溶性人核苷酸酶获得的相似结果进行比较，该结果是由我们的实验室新发现的，该酶是与在流血昆虫的唾液中发现的核苷酸序列相关的，但与entpdase无关。该核苷酸酶的研究很重要，原因有两个。首先，它代表了开发治疗可溶性核苷酸酶的第二个潜在治疗途径。其次，它将证明如何使用两种不同的蛋白质主要结构来执行相同的酶促任务，从而有助于设计更有效的可溶性可溶性核苷酸酶。