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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 我们建议锌K-边缘和EXAFS研究，以获得洞察的结构中发现的中性蛋白酶（NPR，33 kDa）从B的锌活性位点。解淀粉酶Npr属于金属肽酶MA族和M4家族，其中嗜热菌蛋白酶（EC 3.4.24.27）是代表性酶。 Npr，沿着碱性蛋白酶（丝氨酸蛋白酶），代表一种主要的胞外蛋白水解酶。该酶是单体的，并且需要锌和钙离子的存在以获得催化活性及其稳定性。Npr显示与嗜热菌蛋白酶的序列同源性，其中3D晶体结构是已知的。嗜热菌蛋白酶需要一个单一的催化锌离子和四个结构上重要的钙离子。嗜热菌蛋白酶中的Zn原子由三个蛋白质配体（H143、H147和E167）和一个水分子四面体配位。使用嗜热菌蛋白酶晶体数据的Npr结构建模确定了一个Zn和三到四个Ca结合位点。然而，通过电感耦合等离子体质谱法（ICP-MS）对纯化的NPR进行的痕量金属分析显示每个蛋白质分子有两个Zn和两个Ca离子。有先例的第二个锌原子在基质金属蛋白酶和潜在的双核锌网站。例如，据报道基质溶解素-1、明胶酶A和基质溶解素具有结合的两个Zn原子。只有一个Zn原子被认为是催化重要的，而第二个Zn原子起着结构作用。 报道了嗜热菌蛋白酶的第二Zn离子结合的另一个实例。在过量Zn的存在下，第二个Zn离子，被认为是抑制性的，被发现与活性位点附近的酪氨酸（Y158）、组氨酸（H232）和水分子配位，尽管占有率较低（~50%）。 由于锌在光谱上是沉默的，XAS是探测Npr活性位点的独特方法。将在添加一当量和两当量锌的情况下获得Npr的数据，以确定添加第二当量后的结构变化。

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.