Modulation of Streptomyces leucine aminopeptidase by calcium -: Identification and functional analysis of key residues in activation and stabilization by calcium
Modulation of Streptomyces leucine aminopeptidase by calcium -: Identification and functional analysis of key residues in activation and stabilization by calcium
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DOI：
10.1074/jbc.m509025200
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发表时间：
2006-03-03
影响因子：
4.8
通讯作者：
Hatanaka, T
中科院分区：
文献类型：
--
作者：
Arima, J;Uesugi, Y;Hatanaka, T
灰色链霉菌亮氨酸氨肽酶 (SGAP) 的活性位点有两个锌原子，作为了解其他金属肽酶的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。在我们前期的研究中，我们发现了隔状链霉菌分泌的一种非钙调节的亮氨酸氨肽酶，其一级结构与SGAP有71%的一致性。在这项研究中，我们通过使用体内DNA改组系统和几种突变酶通过定点诱变构建了SGAP和S. septatus氨肽酶的嵌合体，以鉴定钙调节中的关键残基。我们鉴定了与 SGAP 激活和钙稳定相关的 SGAP 关键残基 Asp-173 和 Asp-174。我们还表明，已知的钙结合位点由 SGAP 的 Asp-3、Ile-4、Asp-262 和 Asp-266 组成，仅有助于钙对 SGAP 的稳定。此外，我们还发现了一个重要的残基 Glu-196，它与 Asp-173、Asp-174 和钙协同作用，以提高 SGAP 的催化活性。
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.