Greenblatt H M, Almog O, Maras B, Spungin-Bialik A, Barra D, Blumberg S, Shoham G
Department of Inorganic Chemistry, Hebrew University of Jerusalem, Israel.
J Mol Biol. 1997 Feb 7;265(5):620-36. doi: 10.1006/jmbi.1996.0729.
The X-ray crystal structure of the enzyme Streptomyces griseus aminopeptidase (SGAP) has been determined in its double zinc form to 1.75 A resolution, in its apo-enzyme from (zinc removed) to 2.1 A resolution, and as a mercury replaced derivative to 2.1 A resolution. The structure solution was achieved by single isomorphous replacement with phasing from anomalous scattering (SIRAS), followed by density modification with histogram matching. The protein consists of a central beta-sheet made up of eight parallel and antiparallel strands, surrounded by helices on either side. The active site is located at the carbonyl ends of two middle strands of the beta-sheet region. Two sections of the chain that could not be traced were Glu196 to Arg202, which borders the active site, and the final seven C-terminal residues starting with Gly278. The active site contains two zinc cations, each with similar ligands, at a distance of 3.6 A from each other. An unknown molecule appears to be bound to both zinc ions in the active site at partial occupancy and has been modelled as a phosphate ion. A calcium binding site has also been identified, consistent with the observations that calcium modulates the activity of the enzyme, and increases its heat stability. The mechanism by which the calcium cation modulates enzyme activity is not apparent, since the location of the calcium binding site is approximately 25 A distant from the active site zinc ions. Comparison of the structure of SGAP to other known aminopeptidases shows that the enzyme is most similar to Aeromonas proteolytica aminopeptidase (AAP). Both enzymes share a similar topology, although the overall sequence identity is very low (24% in aligned regions). The coordination of the two active site zinc cations in SGAP resembles that of AAP. These two microbial enzymes differ from bovine lens leucine aminopeptidase (LAP) in both overall structure and in coordination of the two zinc ions.
灰色链霉菌氨肽酶（SGAP）的X射线晶体结构已被确定，其双锌形式的分辨率为1.75埃，脱辅基酶形式（去除锌）的分辨率为2.1埃，汞取代衍生物的分辨率为2.1埃。结构解析通过单对同晶置换并利用反常散射进行相位确定（SIRAS），随后通过直方图匹配进行密度修正来实现。该蛋白质由一个由八条平行和反平行链组成的中央β折叠片层构成，两侧环绕着螺旋。活性位点位于β折叠片层区域两条中间链的羰基末端。无法追踪的两段链分别是与活性位点相邻的Glu196至Arg202，以及从Gly278开始的最后七个C末端残基。活性位点包含两个锌阳离子，每个锌阳离子具有相似的配体，彼此相距3.6埃。一个未知分子似乎以部分占有率结合到活性位点的两个锌离子上，并被模拟为磷酸根离子。还鉴定出一个钙结合位点，这与钙调节该酶活性并提高其热稳定性的观察结果一致。钙阳离子调节酶活性的机制尚不明显，因为钙结合位点的位置距离活性位点锌离子约25埃。将SGAP的结构与其他已知氨肽酶进行比较表明，该酶与解蛋白气单胞菌氨肽酶（AAP）最为相似。尽管整体序列同一性非常低（比对区域为24%），但两种酶具有相似的拓扑结构。SGAP中两个活性位点锌阳离子的配位情况与AAP相似。这两种微生物酶在整体结构以及两个锌离子的配位方面均与牛晶状体亮氨酸氨肽酶（LAP）不同。
