QM/MM Simulation
(B3LYP) of the RNase A Cleavage-Transesterification
Reaction Supports a Triester A<sub>N</sub> + D<sub>N</sub> Associative
Mechanism with an O2′ H Internal Proton Transfer
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Abstract
The
mechanism of the backbone cleavage-transesterification step
of the RNase A enzyme remains controversial even after 60 years of
study. We report quantum mechanics/molecule mechanics (QM/MM) free
energy calculations for two optimized reaction paths based on an analysis
of all structural data and identified by a search for reaction coordinates
using a reliable quantum chemistry method (B3LYP), equilibrated structural
optimizations, and free energy estimations. Both paths are initiated
by nucleophilic attack of the ribose O2′ oxygen on the neighboring
diester phosphate bond, and both reach the same product state (PS)
(a O3′–O2′ cyclic phosphate and a O5′
hydroxyl terminated fragment). Path 1, resembles the widely accepted
dianionic transition-state (TS) general acid (His119)/base (His12)
classical mechanism. However, this path has a barrier (25 kcal/mol)
higher than that of the rate-limiting hydrolysis step and a very loose
TS. In Path 2, the proton initially coordinating the O2′ migrates
to the nonbridging O1P in the initial reaction path rather than directly
to the general base resulting in a triester (substrate as base) A<sub>N</sub> + D<sub>N</sub> mechanism with a monoanionic weakly stable
intermediate. The structures in the transition region are associative
with low barriers (TS1 10, TS2 7.5 kcal/mol). The Path 2 mechanism
is consistent with the many results from enzyme and buffer catalyzed
and uncatalyzed analog reactions and leads to a PS consistent with
the reactive state for the following hydrolysis step. The differences
between the consistently estimated barriers in Path 1 and 2 lead to
a 10<sup>11</sup> difference in rate strongly supporting the less
accepted triester mechanism