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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们提出Zn K-edge和EXAFS研究，以深入了解B. amyloliquefaciens中性蛋白酶（Npr, 33 kDa）锌活性位点的结构。Npr属于金属肽酶MA族和M4家族，其中热溶酶（EC 3.4.24.27）为代表酶。Npr与碱性蛋白酶（丝氨酸蛋白酶）一起，是一种主要的细胞外蛋白水解酶。该酶为单体，其催化活性和稳定性需要锌离子和钙离子的存在。Npr显示序列同源的热溶酶，其中三维晶体结构是已知的。热溶酶需要一个催化锌离子和四个结构重要的钙离子。热溶酶中的锌原子是由三个蛋白质配体（H143、H147和E167）和一个水分子组成的四面体配位。利用热溶素晶体数据对Npr进行结构建模，假设定义了1个Zn和3 ~ 4个Ca结合位点。然而，用电感耦合等离子体质谱（ICP-MS）对纯化后的Npr进行了痕量金属分析，发现每个蛋白质分子含有2个Zn和2个Ca离子。在基质金属蛋白酶中有第二个锌原子存在的先例，可能存在双核锌位点。例如，据报道，基质溶酶-1、明胶酶A和基质溶酶有两个锌原子结合。只有一个锌原子被认为是催化重要的，而第二个锌原子起结构作用。第二种锌离子结合的另一个例子报道了热溶酶。在过量Zn存在的情况下，第二个Zn离子被认为具有抑制作用，被发现与酪氨酸（Y158）、组氨酸（H232）和活性位点附近的一个水分子配位，尽管占据率低（~50%）。由于锌在光谱上是沉默的，XAS是一种独特的探测Npr活性位点的方法。数据将在Npr上获得，同时添加一种和两种锌的等价物，以确定添加第二种等价物后的结构变化。

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.