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The mechanistic model evinced by these simulations is consistent with a
considerable body of experimental work, including (1) thio/rescue effect
experiments
63,92
that support a mechanism in which a single metal ion bound
at the C-site in the ground state acquires an additional interaction with the
scissile phosphate in proceeding to the transition state; (2) kinetic studies,
96
photocrosslinking experiments,
119
and mutational data
61,90
that implicate
G8 and G12 as possible general acid and base, respectively; and (3) recent
metal ion titrations suggesting that the p
K
a
of the general acid is down-shifted
by around 4-7 p
K
a
units in a metal ion-dependent manner, correlated with
the metal ion p
K
a
,
120
and that divalent metal ions may play a specific chemical
role in catalysis.
120,121
The direct coordination of Mg
2
þ
and the 2
0
OH of G8
has been confirmed experimentally.
60
Although our simulation results are consistent with most available exper-
imental evidence, there remain caveats that have not yet been fully resolved.
The present work is suggestive that a bound divalent metal ion at the C-site
migrates to the B-site between the A9 and scissile phosphates in proceeding
to the transition state (one metal ion mechanism). However, at this point,
one cannot fully discount an alternate mechanismwhereby there is no direct
participation of a metal ion at the scissile phosphate in the transition state (no
metal ion mechanism), or a mechanism whereby the metal ion at the C-site
does not migrate, but rather the scissile phosphate acquires an additional
metal ion interaction in proceeding to the transition state (the two metal
ion mechanism). In order to fully explore these alternate mechanistic scenar-
ios, full free-energy profiles of the chemical reaction steps are necessary, as
summarized in the next section.
3. FINDING THE CATALYTIC REACTION PATH THROUGH
MULTIDIMENSIONAL FREE-ENERGY SURFACES
In the previous section, we showed that the Mg
2
þ
metal ion may
migrate from the C-site to the B-site when C17:O2
0
is activated
(deprotonated), and may be directly involved in the general acid step by
coordinating with the general acid, the 2
0
OH group of G8. Two 1 ns
QM/MM simulations on fixed reaction coordinate points, representing
early and LTSs, support such a picture.
75,76
In this section, we report our
effort to further extend our QM/MM calculations to obtain the free-energy
profile along the relevant reaction coordinates to explore the proposed metal
ion-assisted phosphoryl transfer and general acid catalysis mechanism in
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