Arima Jiro, Uesugi Yoshiko, Uraji Misugi, Yatsushiro Shouki, Tsuboi Seiji, Iwabuchi Masaki, Hatanaka Tadashi
Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
J Biol Chem. 2006 Mar 3;281(9):5885-94. doi: 10.1074/jbc.M509025200. Epub 2005 Dec 28.
Streptomyces griseus leucine aminopeptidase (SGAP), which has two zinc atoms in its active site, is clinically important as a model for understanding the structure and mechanism of action of other metallopeptidases. SGAP is a calcium-activated and calcium-stabilized enzyme, and its activation by calcium correlates with substrate specificity. In our previous study, we found a non-calcium-modulated leucine aminopeptidase secreted by Streptomyces septatus, the primary structure of which showed 71% identity with SGAP. In this study, we constructed chimeras of SGAP and S. septatus aminopeptidase by using an in vivo DNA shuffling system and several mutant enzymes by site-directed mutagenesis to identify the key residues in this modulation by calcium. We identified the key residues Asp-173 and Asp-174 of SGAP associated with both SGAP activation and stabilization by calcium. We also showed that the known calcium-binding site, which is composed of Asp-3, Ile-4, Asp-262, and Asp-266 of SGAP, only contributes to SGAP stabilization by calcium. Furthermore, we identified an important residue, Glu-196, that functions in cooperation with Asp-173, Asp-174, and calcium to increase the catalytic activity of SGAP.
灰色链霉菌亮氨酸氨肽酶（SGAP）在其活性位点含有两个锌原子，作为理解其他金属肽酶结构和作用机制的模型，在临床上具有重要意义。SGAP是一种钙激活且钙稳定的酶，其被钙激活与底物特异性相关。在我们之前的研究中，我们发现了一种由栖土链霉菌分泌的非钙调节亮氨酸氨肽酶，其一级结构与SGAP有71%的同一性。在本研究中，我们利用体内DNA改组系统构建了SGAP和栖土链霉菌氨肽酶的嵌合体，并通过定点诱变构建了几种突变酶，以确定钙调节过程中的关键残基。我们确定了SGAP的关键残基Asp-173和Asp-174，它们与SGAP被钙激活和稳定均相关。我们还表明，由SGAP的Asp-3、Ile-4、Asp-262和Asp-266组成的已知钙结合位点仅有助于SGAP被钙稳定。此外，我们确定了一个重要残基Glu-196，它与Asp-173、Asp-174和钙协同作用以提高SGAP的催化活性。
