PROTEASE SPECIFICITY AND REGULATION PROTEIN ENGINEERING

蛋白酶特异性和调控蛋白质工程

• 批准号: 8168792
• 负责人: Enrico Di Cera
• 金额: $ 0.01万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168792
• 关键词: Active Sites; Architecture; Binding; Binding Sites; Cells; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Equilibrium; Evolution; Fluorescence; Funding; Grant; Human; Institution; Life; Measurement; Molecular Conformation; Peptide Hydrolases; Phase; Printing; Protein Engineering; Prothrombin; Regulation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Source; Specificity; Structure; Thrombin; United States National Institutes of Health; inhibitor/antagonist; meizothrombin; phenylalanyl-prolyl-arginine-chloromethyl ketone

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na(+)-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 A resolution. The structure reveals a Na(+) binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na(+) binding to meizothrombin desF1 document a slow phase of fluorescence change with a k (obs) decreasing hyperbolically with increasing [Na(+)], consistent with the existence of three conformations in equilibrium, E*, E and E:Na(+), as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation. Papaconstantinou, M.E., Gandhi, P.S., Chen, Z., Bah, A. and Di Cera, E. Na(+) Binding to meizothrombin desF1. Cell Mol Life Sci. (E-pub ahead of print.) (2008). Page, M.J. and Di Cera, E. Evolution of peptidase diversity. J Biol Chem (E-pub ahead of print.) (2008).

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 凝血酶原是在 R320 处单次裂解凝血酶原以分离 A 链和 B 链而产生的生理活性中间体。最近的证据表明，与凝血酶一样，酶促凝血酶是一种 Na(+) 激活的酶。在这项研究中，我们展示了在活性位点抑制剂 PPACK 存在下以 2.1 A 分辨率解析的人酶凝血酶 desF1 的第一个 X 射线晶体结构。该结构揭示了一个 Na(+) 结合位点，其结构实际上与人类凝血酶相同。 Na(+) 与酶促凝血酶 desF1 结合的停流测量记录了荧光变化的缓慢阶段，其中 k (obs) 随着 [Na(+)] 的增加呈双曲线下降，这与平衡状态下 E*​​、E 三种构象的存在一致和E:Na(+)，对于人凝血酶。酶促凝血酶以多种构象存在的证据为凝血酶原激活机制的研究提供了有价值的新信息。 Papaconstantinou, M.E.、Gandhi, P.S.、Chen, Z.、Bah, A. 和 Di Cera, E. Na(+) 与酶促凝血酶 desF1 结合。细胞分子生命科学。 （印刷前的电子出版。）（2008）。 Page, M.J. 和 Di Cera, E. 肽酶多样性的进化。 J Biol Chem（电子版，印刷前）（2008）。