Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations

Abstract

The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [BakerE. N.BakerE. N.7003158J. Mol. Biol.1980141441484; BakerE. N.BakerE. N.859183J. Mol. Biol.1977111207210; DrenthJ.DrenthJ.952885Biochemistry19761537313738; MenardR.MenardR.J. Cell. Biochem.1994137; PolgarL.PolgarL.689035Eur. J. Biochem.197888513521; StorerA. C.StorerA. C.7845227Methods Enzymol.1994244486500. 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.

Structural analyses of Arabidopsis thaliana legumain γ reveal differential recognition and processing of proteolysis and ligation substrates

“…Conversely, AtLEGβ may be a superior ligase over AtLEGγ for substrates with P2′ residues optimized for AtLEGβ's amphiphilic S2′ site. These findings are in perfect agreement with a computational report on human legumain-mediated transpeptidation, which was only possible if water was excluded from the active site ( 38 ). Finally we note that the proposed water displacement model is consistent with the reportedly low proteolytic activity of butelase ( 15 ) as well as the here observed ≈5000-fold decreased proteolytic activity of AtLEGγ as compared with human legumain ( Fig.…”

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study

“…From our FESs it is clear that the first step of the reaction (activation of the cysteine and the activation requires a minor energy expense. This results to be a peculiarity of this artificial protease, since in the natural one generally, the nucleophilic attack is the rate-limiting steps [ 27 ].…”

Unraveling the SARS-CoV-2 Main Protease Mechanism Using Multiscale DFT/MM Methods

“…In order to explore other possible mechanisms 27 we also studied a reaction path that does not involve the formation of an ion pair, although the associated free energy barrier is considerably higher and incompatible with the experimental rate constant (see Figure S3).…”