On the Nature of the Enzyme–Substrate Complex and the Reaction Mechanism in Human Arginase I. A Combined Molecular Dynamics and QM/MM Study

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.