Abstract
The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [BakerE. N.J. Mol. Biol.1980, 141, 441−4847003158; BakerE. N.; J. Mol. Biol.1977, 111, 207–210859183; DrenthJ.; Biochemistry1976, 15, 3731–3738952885; MenardR.; J. Cell. Biochem.1994, 137; PolgarL.Eur. J. Biochem.1978, 88, 513–521689035; StorerA. C.; Methods Enzymol.1994, 244, 486–5007845227. 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.
Highlights
The most common cysteine proteases are papain, cathepsin, and caspases, which can be found in a series of living organisms[1−7] and play significant roles in proteolytic signaling
Due to the fact that in the cystatin− legumain complex the NE(His148)−SG(Cys189) distance is over 6 Å and in prolegumain these residues are only 4.14 Å from each other, first we supposed that the proton shuttle between them might occur, before the substrate enters the active site, and as soon as the catalytic cycle is completed and the substrate leaves the pocket, the zwitterion regenerates
We present mechanistic details of the enzymatic protease and ligase activity of human legumain using hybrid quantum mechanics/molecular mechanics (QM/MM) methods at the density functional theory (DFT)/b3lyp level of theory
Summary
The most common cysteine proteases are papain, cathepsin, and caspases, which can be found in a series of living organisms[1−7] and play significant roles in proteolytic signaling. The most relevant theoretical work with respect to our studies are the investigations of Sulpizi et al.,[42] who applied DFT-based QM/ MM methods to calculate the hydrolysis of the acyl enzyme complex for caspase-3 starting from a covalently bound inhibitor Their calculations suggest that the attack of the nucleophilic water molecule (second step of the cleavage) leads to a geminal diol intermediate and shows thereby a remarkable discrepancy between caspases and papain. Miscione et al.[43] performed DFT calculations in the gas phase to study thioester formation for caspase-7 starting from a covalently bound inhibitor complex Their model was built up from fragments of the active site residues and the Ac-DEVD inhibitor. We would like to emphasize that detailed experimental work is not part of the present paper
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