X-RAY ABSORPTION SPECTROSCOPIC STUDIES OF NATURAL PROTEASE (NPRE)

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。 我们提出了锌K-EDGE和EXAFS研究，以深入了解淀粉液化芽孢杆菌中性蛋白酶(NPR，33 kDa)中锌活性部位的结构。NPR属于金属多肽酶MA家族和M4家族，其中以嗜热分解酶(EC 3.4.24.27)为代表。NPR与碱性蛋白水解酶(丝氨酸蛋白酶)一起，是一种主要的胞外蛋白水解酶。该酶是单体酶，需要锌和钙离子的存在才能保证其催化活性和稳定性。NPR显示了与已知3D晶体结构的热裂解蛋白的序列同源性。热裂解酶需要一个催化的锌离子和四个结构上重要的钙离子。热裂解酶中的锌原子是由三个蛋白质配体(H143、H147和E167)和一个水分子四面体配位的。利用热裂解蛋白的晶体数据对NPR的结构进行建模，推测定义了一个锌和三到四个钙结合部位。然而，通过电感耦合等离子体质谱(ICP-MS)对纯化的NPR进行微量金属分析，发现每个蛋白质分子有两个锌离子和两个钙离子。在基质金属蛋白酶中有第二个锌原子和潜在的双核锌位点的先例。例如，据报道，基质分解素-1、明胶酶A和基质分解素有两个锌原子结合。然而，只有一个锌原子被认为是催化重要的，而第二个锌原子起着结构作用。第二个锌离子结合的另一个例子被报道为热裂解酶。在过量的锌存在下，第二个锌离子被认为是抑制的，被发现与活性中心附近的酪氨酸(Y158)、组氨酸(H232)和水分子配位，尽管占有率很低(~50%)。由于锌在光谱上是无声的，XAS是探测NPR活性中心的唯一方法。将获得关于NPR的数据，其中添加了锌的一个和两个当量，以确定添加第二个当量后的结构变化。

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. We propose Zn K-edge and EXAFS studies in order to obtain insight into the structure of the Zinc active site found in neutral protease (Npr, 33 kDa) from B. amyloliquefaciens. Npr belongs to a metallopeptidases MA clan and M4 family in which thermolysin (EC 3.4.24.27) is a representative enzyme. Npr, along with alkaline protease (serine protease), represents a major extracellular proteolytic enzyme. The enzyme is monomeric and requires presence of zinc and calcium ions for the catalytic activity and its stability. Npr shows sequence homology to thermolysin in which the 3D crystal structure is known. Thermolysin requires a single catalytic Zn ion and four structurally important Ca ions. The Zn atom in thermolysin is tetrahedrally coordinated by three protein ligands (H143, H147, and E167) and a water molecule. Structure modeling of Npr using the thermolysin crystal data putatively defined one Zn and three to four Ca binding sites. However, a trace metal analysis of purified Npr by the inductively coupled plasma mass spectroscopy (ICP-MS) showed two Zn and two Ca ions per protein molecule. There are precedents for the second Zn atom in matrix metalloproteinase and potentially a binuclear Zinc site. For example, stromelysin-1, gelatinase A, and matrilysin are reported to have two Zn atoms bound. Only one Zn atom is thought to be catalytically important whereas the second Zn atom plays a structural role, however. Another example of the second Zn ion binding is reported for the thermolysin. In the presence of excess Zn, the second Zn ion, proposed to be inhibitory, is found coordinated to a tyrosine (Y158), histidine (H232), and a water molecule in the vicinity of the active site, albeit at a low occupancy (~50%). As Zinc is spectroscopically silent, XAS is a unique method for probing the Npr active site. Data will be obtained on Npr with the addition of both one and two equivalents of zinc, to determine the structural changes upon addition of the second equivalent.