PROTEASE SPECIFICITY AND REGULATION PROTEIN ENGINEERING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 美索托凝血酶是一种生理活性中间体，由凝血酶原在R320处的一次裂解产生，以分离A链和B链。最近的证据表明，甲硫凝血酶和凝血酶一样，是一种Na(+)激活的酶。在这项研究中，我们提出了在2.1A分辨率下，在活性位点抑制剂PPACK存在下解决的人甲硫托品DesF1的第一个X射线晶体结构。该结构揭示了一个Na(+)结合部位，其结构实际上与人凝血酶的结构相同。与人凝血酶一样，Na(+)与甲硫凝血酶desF1结合的停流测量表明，随着[Na(+)]的增加，k(Obs)呈双曲线下降，这与E*、E和E：Na(+)三种平衡构象的存在一致。甲硫咪唑凝血酶以多种构象存在的证据为研究凝血酶原激活机制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meizothrobin desF1结合。细胞摩尔生命科学。(印刷前的电子酒吧)(2008)。 多肽酶多样性的进化.北京：科学出版社.《生物化学》(印刷前的电子酒吧)(2008)。

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).