A Multiple Proton Transfer Mechanism for the Charging Step of the Aminoacylation Reaction at the Active Site of Aspartyl tRNA Synthetase

Aspartyl-tRNA synthetase catalyzes the attachment of
aspartic acid
to its cognate tRNA by the aminoacylation reaction during the initiation
of the protein biosynthesis process. In the second step of the aminoacylation
reaction, known as the charging step, the aspartate moiety is transferred
from aspartyl-adenylate to the 3′-OH of A76 of tRNA through
a proton transfer process. We have investigated different pathways
for the charging step through three separate QM/MM simulations combined
with the enhanced sampling method of well-sliced metadynamics and
found out the most feasible pathway for the reaction at the active
site of the enzyme. In the charging reaction, both the phosphate group
and the ammonium group after deprotonation can potentially act as
a base for proton transfer in the substrate-assisted mechanism. We
have considered three possible mechanisms involving different pathways
of proton transfer, and only one of them is determined to be enzymatically
feasible. The free energy landscape along reaction coordinates where
the phosphate group acts as the general base showed that, in the absence
of water, the barrier height is 52.6 kcal/mol. The free energy barrier
is reduced to 39.7 kcal/mol when the active site water molecules are
also treated quantum mechanically, thus allowing a water mediated
proton transfer. The charging reaction involving the ammonium group
of the aspartyl adenylate is found to follow a path where first a
proton from the ammonium group moves to a water in the vicinity forming
a hydronium ion (H3O+) and NH2 group.
The hydronium ion subsequently passes the proton to the Asp233 residue,
thus minimizing the chance of back proton transfer from hydronium
to the NH2 group. The neutral NH2 group subsequently
takes the proton from the O3′ of A76 with a free energy barrier
of 10.7 kcal/mol. In the next step, the deprotonated O3′ makes
a nucleophilic attack to the carbonyl carbon forming a tetrahedral
transition state with a free energy barrier of 24.8 kcal/mol. Thus,
the present work shows that the charging step proceeds through a multiple
proton transfer mechanism where the amino group formed after deprotonation
acts as the base to capture a proton from O3′ of A76 rather
than the phosphate group. The current study also shows the important
role played by Asp233 in the proton transfer process.