PROTEASE SPECIFICITY AND REGULATION PROTEIN ENGINEERING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 甲唑凝血酶是通过凝血酶原在R320处单次裂解以分离A和B链而产生的生理活性中间体。最近的证据表明，美佐凝血酶，像凝血酶一样，是一种Na（+）激活酶。在这项研究中，我们提出了第一个X-射线晶体结构的人meizothrobin desF 1解决的活性位点抑制剂PPACK的存在下，在2.1 A的分辨率。该结构揭示了Na（+）结合位点，其结构与人凝血酶的结构几乎相同。Na（+）与meizothrobin desF 1结合的停流测量记录了荧光变化的缓慢阶段，k（obs）随着[Na（+）]的增加呈双曲线下降，与人凝血酶中存在的三种平衡构象E*、E和E：Na（+）一致。甲藻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.，甘地，附言，陈志，Bah，A.和Di Cera，E. Na（+）与甲藻凝血酶desF 1结合。细胞分子生命科学（电子出版先于印刷。）（2008年）。 Page，M.J.和Di Cera，E.肽酶多样性的进化。J Biol Chem（电子出版，印刷前）（2008年）。

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).