Gilboa R, Spungin-Bialik A, Wohlfahrt G, Schomburg D, Blumberg S, Shoham G
Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem, Israel.
Proteins. 2001 Sep 1;44(4):490-504. doi: 10.1002/prot.1115.
Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.
灰色链霉菌氨肽酶（SGAP）是一种双锌外肽酶，对大的疏水性氨基末端残基具有高度偏好。它是一种分子量相对较低（30 kDa）的单体，具有热稳定性，表现出高效的催化周转，其活性受钙离子调节。小尺寸、高活性和热稳定性使SGAP成为各种生物技术应用中极具吸引力的酶，其中包括重组DNA蛋白和融合蛋白产物的加工。发现几种游离氨基酸，如苯丙氨酸、亮氨酸和蛋氨酸，可作为SGAP的弱抑制剂，因此被选用于结构研究。这些抑制剂可能被视为产物类似物，因为在正常酶促反应中获得的产物之一是底物的切割氨基末端氨基酸。当前的研究包括对SGAP与蛋氨酸（分辨率为1.53 Å）、亮氨酸（分辨率为1.70 Å）和苯丙氨酸（分辨率为1.80 Å）形成的复合物进行X射线晶体学分析。这三种高分辨率结构已被用于全面表征SGAP活性位点，并确定参与酶-底物和酶-产物相互作用的酶的一些官能团。底物末端胺基的独特结合位点（包括Glu131和Asp160的侧链以及Arg202的羰基）在催化反应的底物和产物的结合与定向中起着重要作用。这些研究还表明，Glu131和Tyr246直接参与酶的催化机制。这两个残基似乎对底物结合和定向以及酶-底物复合物的四面体过渡态的稳定都很重要。特别提出Glu131在催化过程中通过促进锌结合的水/氢氧根对底物羰基碳的亲核攻击而作为通用碱发挥作用。将三种SGAP复合物的结构与三种相关氨肽酶的最新结构进行比较：解蛋白气单胞菌氨肽酶（AAP）、亮氨酸氨肽酶（LAP）和蛋氨酸氨肽酶（MAP）以及它们与相应抑制剂和类似物形成的复合物。这些结构结果已用于沿反应坐标模拟几种物种，并用于提出由SGAP催化的蛋白水解反应的通用方案。
