Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations.
Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations.
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DOI：
10.1021/acscatal.7b01505
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发表时间：
2017-09-01
期刊：
影响因子：
12.9
通讯作者：
Brandstetter H
中科院分区：
文献类型：
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作者：
Elsässer B;Zauner FB;Messner J;Soh WT;Dall E;Brandstetter H
半胱氨酸蛋白酶 Legumain 在天冬酰胺之后以及在天冬氨酸之后在更酸性的条件下以高特异性水解肽键 [, , −; ; , , –; ; , , –; ; , ; , , –; ; , , –.值得注意的是，legumain 还表现出在 pH > 5.5 时普遍存在的连接酶活性。原子反应机制（包括其 pH 依赖性）仅被部分了解。在这里，我们提出了基于密度泛函理论（DFT）的量子力学/分子力学（QM/MM）研究人类legumain在溶液中的两种活性的详细反应机制。与其他木瓜蛋白酶样蛋白酶的情况相比，我们的计算表明，活性位点 Cys189 必须以质子化状态存在，才能进行有效的亲核攻击并同时断裂肽键，这与 legumain 催化裂解的实验 pH 曲线一致。生成的硫酯中间体 (INT1) 通过水对硫酯的攻击转化为第二种中间体，二醇 (INT2)，通过 Cys189 夺取质子而释放。令人惊讶的是，我们发现连接并不是蛋白水解的完全相反过程，而是可以通过两种不同的途径进行。尽管转肽途径涉及硫酯 (INT1) 的氨解，但在 pH 6 时发现了不依赖于半胱氨酸、组氨酸辅助的连接途径。鉴于legumain在免疫、癌症和神经退行性疾病中的重要作用，我们的研究结果为这些领域的靶向药物设计开辟了可能性。
The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [, , −; ; , , –; ; , , –; ; , ; , , –; ; , , –. Remarkably, legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given legumain’s important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields.
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影响因子：
16.8
作者：
Koehnke, Jesko;Bent, Andrew;Houssen, Wael E.;Zollman, David;Morawitz, Falk;Shirran, Sally;Vendome, Jeremie;Nneoyiegbe, Ada F.;Trembleau, Laurent;Botting, Catherine H.;Smith, Margaret C. M.;Jaspars, Marcel;Naismith, James H.
通讯作者：
Naismith, James H.
影响因子：
2.9
作者：
GOPALAN, P;DUFRESNE, MJ;WARNER, AH
通讯作者：
WARNER, AH
影响因子：
5.6
作者：
BAKER, EN
通讯作者：
BAKER, EN
影响因子：
--
作者：
Bernath-Levin, Kalia;Nelson, Clark;Mylne, Joshua S.
通讯作者：
Mylne, Joshua S.
人豆蛋白酶中依赖天冬氨酸的肽连接酶的结构和机制。
DOI：
10.1002/anie.201409135
发表时间：
2015-03-02
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Dall E;Fegg JC;Briza P;Brandstetter H
通讯作者：
Brandstetter H