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Figure 4.
Figure 4. Active site cavity and the binding of AMPCPP,
pantoate, and pantoyl adenylate. (A) A stereo view of the active
site cavity of subunit A of the complex with both AMPCPP and
pantoate. The substrates (both with partial occupancy) are shown
as ball-and-stick models. The active site cavity is surrounded
by ß2-loop-  2, ß7-loop,
ß6-loop- 6,
3[10]5'-loop- 5, and
ß3-loop-3[10]3- 3'-loop, and
covered by 3[10]7 and the ß-sheet of C-terminal domain. Residues
around helix 3' (shown in
cyan) are disordered in subunit B, which has a fully occupied
AMPCPP and a glycerol molecule in the active site. (B) A section
of the initial difference electron density map (Fo - Fc) in the
active site of subunit B superimposed on the refined model,
calculated at 1.7 Å and contoured at the 2  level. Side
chains of Lys160, Ser196, and Arg198 have moved relative to
those in the apo enzyme to interact with the phosphate groups,
and thus also have positive initial difference electron density.
The electron density figures are prepared with PYMOL (DeLano
2002). (C) Detailed binding interactions between AMPCPP (shown
with carbon atoms in gold) and protein active site residues of
subunit B. The Mg2+ ion is shown as a yellow sphere, and water
molecules are shown as red spheres. Hydrogen bonds between
AMPCPP and protein atoms, and some water-mediated hydrogen bonds
are shown as dashed lines. A glycerol molecule found next to the
-phosphate of
AMPCPP, at the pantoate binding site, is also shown. (D) A
section of the initial difference electron density (Fo - Fc)
around the bound pantoate molecule in the active site of subunit
A of the pantoate-ß-alanine complex (data set 7) shows that
pantoate is very well ordered with full occupancy. The nearby
residues did not move relative to those of the apo enzyme, and
therefore did not have initial difference density. The electron
density was calculated at 1.7 Å and contoured at 2 . (E) The
pantoate molecule (shown in gold for the carbon atoms) is
tightly bound and fits snugly in its binding site. Two glutamine
side chains form hydrogen bonds to the hydroxyl groups and one
carboxyl oxygen of the pantoate. The two methyl groups and the
hydrophobic side of pantoate interact with the side chains of
Pro38, Met40, and Phe157. (F) A section of the initial
difference electron density (Fo - Fc) around the bound pantoyl
adenylate molecule in the active site of subunit B of the
intermediate complex (data set 6) shows that intermediate is
very well ordered with full occupancy. The electron density was
calculated at 1.7 Å and contoured at 2 . (G) The
pantoyl adenylate molecule (shown with carbon atoms in gold) is
tightly bound and fits snugly in the active site cavity. The
adenosine and pantoyl groups are at identical positions as those
in the AMPCPP complex and pantoate complex, respectively, and
have identical interactions with the active site residues.
However, the -phosphate
moved down to have a covalent bond to the pantoate, which allows
the phosphate group to have a hydrogen bond to the amide
nitrogen of Met40.
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