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PDBsum entry 2caq
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References listed in PDB file
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Key reference
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Title
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Probing the mechanism of gsh activation in schistosoma haematobium glutathione-S-Transferase by site-Directed mutagenesis and X-Ray crystallography.
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Authors
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P.Baiocco,
L.J.Gourlay,
F.Angelucci,
J.Fontaine,
M.Hervé,
A.E.Miele,
F.Trottein,
M.Brunori,
A.Bellelli.
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Ref.
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J Mol Biol, 2006,
360,
678-689.
[DOI no: ]
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PubMed id
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Abstract
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During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class
Schistosoma haematobium wild-type glutathione-S-transferase is expected to
switch alternately in and out of the reduced glutathione-binding site (G-site).
The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group
of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has
a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the
structural and functional roles of Arg21, we carried out structural studies on
two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic
data for the two Arg21 mutants indicate that only the Tyrout10 conformation is
populated, thereby excluding a role of Arg21 in the stabilisation of the out
conformation. However, Arg21 was confirmed to be catalytically important and
essential for the low pKa of Tyr10. Upon comparison with structural data
generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes,
it was observed that the orientations of Tyr10 and Arg35 are concerted and that,
upon ligand binding, minor rearrangements occur within conserved residues in the
active site loop. These rearrangements are coupled to quaternary rigid-body
movements at the dimer interface and alterations in the localisation and
structural order of the C-terminal domain.
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Figure 1.
Figure 1. The movement of conserved Arg35. The two
alternative positions of Arg35 were observed in the structures
of (a) GTX-bound wtGST showing only the Tyr^in10 conformer and
(b) R21L showing the Tyr^out10 conformer. Arg35 is indicated by
green sticks, whereas other important residues are indicated by
blue sticks. This Figure was generated using ViewerLite 4.2
(Accelrys Inc.).
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Figure 2.
Figure 2. Stereo views of the electron density (2F[o]–F[c]
at 1σ) showing the two conformers of Tyr10 in the G-site. (a)
R21L, showing Tyr^out10 at H-bond distance from Asp33. The new
residue, Leu21 is at a distance of 4 Å from Tyr10.
Tyr^out10 hinders the movement of the Arg35 side-chain, as
displayed in Figure 1(b). (b) GTX-bound wtGST, showing the polar
interaction between the hydroxyl group of the Tyr^in10 and the
sulphur atom of GTX. Tyr10, Arg21, Asp33 and Arg35 are
orientated as shown in Figure 1(a).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
360,
678-689)
copyright 2006.
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