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The reaction mechanism of phosphoryl transfer catalyzed
by UMP/CMP-kinase from Dictyostelium discoideum
was investigated by semiempirical AM1 molecular orbital
computations of an active site model system derived from
crystal structures that contain a transition state analog
or a bisubstrate inhibitor. The computational results suggest
that the nucleoside monophosphate must be protonated for
the forward reaction while it is unprotonated in the presence
of aluminium fluoride, a popular transition state analog
for phosphoryl transfer reactions. Furthermore, a compactification
of the active site model system during the reaction and
for the corresponding complex containing AlF3
was observed. For the active site residues that are part
of the LID domain, conformational flexibility during the
reaction proved to be crucial. On the basis of the calculations,
a concerted phosphoryl transfer mechanism is suggested
that involves the synchronous shift of a proton from the
monophosphate to the transferred PO3-group.
The proposed mechanism is thus analogous to the phosphoryl
transfer mechanism in cAMP-dependent protein kinase that
phosphorylates the hydroxyl groups of serine residues.
The reaction mechanism of the catalytic phosphoryl
transfer of cAMP-dependent protein kinase (cAPK) was investigated
by semi-empirical AM1 molecular orbital computations of
an active site model system derived from the crystal structure
of the catalytic subunit of the enzyme. The activation
barrier is calculated as 20.7 kcal mol−1
and the reaction itself to be exothermic by 12.2 kcal mol−1.
The active site residue Asp166, which was often proposed
to act as a catalytic base, does not accept a proton in
any of the reaction steps. Instead, the hydroxyl hydrogen
of serine is shifted to the simultaneously transferred
phosphate group of ATP. Although the calculated transition
state geometry indicates an associative phosphoryl transfer,
no concentration of negative charge is found. To study
the influence of protein mutations on the reaction mechanism,
we compared two-dimensional energy hypersurfaces of the
protein kinase wild-type model and a corresponding mutant
in which Asp166 was replaced by alanine. Surprisingly,
they show similar energy profiles despite the experimentally
known decrease of catalytic activity for corresponding
mutants. Furthermore, a model structure was examined, where
the charged NH3 group of Lys168 was replaced
by a neutral methyl group. The energetic hypersurface of
this hypothetical mutant shows two possible pathways for
phosphoryl transfer, which both require significantly higher
activation energies than the other systems investigated,
while the energetic stabilization of the reaction product
is similar in all systems. As the position of the amino
acid side chains and the substrate peptide is virtually
unchanged in all model systems, our results suggest that
the exchange of Asp166 by other amino acid is less important
to the phosphoryl transfer itself, but crucial to maintain
the configuration of the active site in vivo. The positively
charged side chain of Lys168, however, is necessary to
stabilize the intermediate reaction states, particularly
the side chain of the substrate peptide.
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