Laboratory of Structural Chemistry and Biology and HAS-ELTE Protein Modeling Group, Institute of Chemistry, Eötvös Loránd University, Pázmány P. sétány 1/A, H-1117 Budapest, Hungary.
J Biol Chem. 2011 Jan 21;286(3):1987-98. doi: 10.1074/jbc.M110.169862. Epub 2010 Nov 16.
Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.
从 Aeropyrum pernix 中提取的酰基氨酰肽酶是一种同二聚体，属于脯氨酰寡肽酶家族。单体亚基由一个水解酶和一个推进器结构域组成。以前的晶体结构测定表明，推进器结构域阻碍了底物进入两个亚基的活性位点。在这里，我们研究了两种突变酶的结构和动力学，其中催化三联体的天冬氨酸被改变为丙氨酸或天冬酰胺。使用不同的底物，我们确定了特异性速率常数、催化限速步骤以及底物和抑制剂结合的 pH 依赖性。催化作用在很大程度上取决于突变的种类和底物的性质。结果根据催化组氨酸侧链的位置变化进行了解释，这在天然和两种突变结构（D524N 和 D524A）的晶体结构测定中得到了证明。出乎意料的是，在同二聚体结构中，只有一个亚基显示出酶的封闭形式。另一个亚基显示出通向催化位点的开放门，从而揭示了控制寡肽酶活性的结构基础。天然酶的开放形式显示出催化三联体处于扭曲的非活性状态。突变影响酶的封闭、活性形式，破坏其催化三联体。我们得出结论，两种形式处于平衡状态，底物通过构象选择机制结合。
