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.
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