On the Nature of the Enzyme–Substrate Complex and the Reaction Mechanism in Human Arginase I. A Combined Molecular Dynamics and QM/MM Study
datasetposted on 17.07.2020, 12:36 by José L. Velázquez-Libera, Julio Caballero, Iñaki Tuñón, Erix W. Hernández-Rodríguez, J. Javier Ruiz-Pernía
We present here a complete detailed theoretical analysis of l-arginine hydrolysis catalyzed by human arginase I (HARGI). Our study combines classical molecular dynamics (MD) simulations of different models for the enzyme–substrate complex and a detailed exploration of the reaction mechanism for the most plausible model using hybrid quantum mechanics/molecular mechanics techniques. Different enzyme–substrate models were built considering that the nucleophile in charge of the initial attack to the substrate could either be a water molecule or a hydroxide anion bridging between the two manganese(II) cations present in the active site. In contrast, for the substrate, we considered four different possibilities: one with the guanidino group of the substrate protonated and three of them where this group is neutral. In this last case, we considered two different tautomeric states and two possible coordination modes with the divalent ions. Our MD simulations revealed that the most stable complexes correspond to those having a hydroxide anion as the nucleophile, while both a protonated and a neutral form of the guanidino group can bind into the active site. Our analysis of the potential energy surface reveals a complex reaction pathway, where the initial attack of the nucleophile is followed by the inversion of the epsilon nitrogen atom of the guanidino group. It produces a reaction intermediate, stabilized by means of an H−π interaction with a histidine residue, that shows high structural similitude with some of the most potent inhibitors known for HARGI (those derived from the 2(S)-amino-6-boronohexanoic acid). Subsequently, a proton is transferred from the nucleophile to the leaving group via Asp128. The final step corresponds to the separation of the two reaction fragments: l-ornithine and urea. Our analysis is shown to be robust by comparison among the results obtained with different initial structures and functionals and also because of the good agreement with the experimental estimations of the activation energy and with mutagenesis analysis. Finally, the analysis of the electronic distribution demonstrates that manganese ions are not involved in charge-transfer processes during the reaction pathway, playing a structural and electrostatic role to stabilize the nucleophile and intermediate states of the reaction.