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PDBsum entry 2gtu
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The enhanced affinity for thiolate anion and activation of enzyme-Bound glutathione is governed by an arginine residue of human mu class glutathione s-Transferases.
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Authors
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Y.V.Patskovsky,
L.N.Patskovska,
I.Listowsky.
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Ref.
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J Biol Chem, 2000,
275,
3296-3304.
[DOI no: ]
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PubMed id
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Abstract
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A series of chimeric human Mu class glutathione S-transferases were designed to
determine mechanisms by which they activate enzyme-bound glutathione (GSH) for
reaction with electrophilic substrates. In view of evidence that the His(107)
residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska,
L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate
Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and
arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and
hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably
associated with enzymes containing an Arg(107) residue include an inverse
dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes
with other residues at that position. Moreover, affinities for GSH thiolate
anion binding are greater for enzymes containing Arg(107))), with K(d) values of
20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained
by steady-state kinetic analyses. Both thermodynamic and kinetic and data
indicate that the Arg(107))) residue is specifically involved in enhancing the
binding affinity of GSH thiolate anion relative to that of the protonated form.
These enzymes therefore, can be more effective at lower GSH concentrations.
Combined mutations indicate that both Arg(107) and Tyr(6) residues are required
for thiolate anion formation and stabilization. The three-dimensional structure
of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that
Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3
A apart), but is distant from the GSH-binding site. However, an alternative
energetically favorable model places the guanidino group 4 A from the sulfur
atom of bound GSH. It is suggested therefore, that in solution, motion of the
positively charged arginine into the catalytic pocket could provide a counter
ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for
the observed greater affinity of enzymes containing Arg(107) for binding of
thiolate anion.
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Figure 1.
Fig. 1. Dependence of kinetic constants k[cat] and
k[cat]/K[m] for the CDNB substrate (A, B, and C) and CDNBA (D,
E, and F) on pH. Data are for the enzyme catalyzed reaction
between GSH and CDNB as described under "Experimental
Procedures." Results for the wild-type recombinant hGSTM2-2 (
 ), and
the following mutants; Y6F (  ), R107H (
 circle ),
R107N (  ), and a
double mutant Y6F,R107H (  ) are
shown.
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Figure 2.
Fig. 2. GST-dependent thiolate anion formation. A,
thiolate anion formation as a function of pH. Results for
wild-type hGSTM2-2 ( ), and
mutants, Y6F ( ), R107H (
circle ),
and R107N ( ) are shown.
A protein concentration of 20 µM and a saturating GSH
concentration of 1.0 m M were used to obtain UV difference
spectra shown in the inset. Difference values of absorbance at
240 nm were plotted versus pH. Inset, difference spectra for the
binary complexes of wild-type hGSTM2-2 (solid line), and Y6F (-
-), and R107N (- - -) mutants with GSH in 100 mM sodium
phosphate buffer, pH 6.8. For each variant, difference spectra
of the indicated forms of the enzyme (20 µM) complexed
with 50 µM GSH or 500 µM GSH for the R107N mutant
are shown. A solution of GSH alone in the same buffer was used
as a reference, and the absorbance of the protein alone in this
spectral range was also corrected to yield the difference
spectra. B, thiolate anion formation as a function of GSH
concentration. Each GST variant (20 µM) was dissolved in
0.1 M sodium phosphate buffer, pH 6.8, and, after addition of
the indicated amounts of GSH the differential absorbance at 240
nm was determined. Enzymes represented are wild-type hGSTM2-2 (
),
hGSTM2-2 mutants R107H ( circle ),
R107N ( ),
hGSTM1a-1a (H107R)(+), and hGSTM4-4 (S107R) (  ).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
3296-3304)
copyright 2000.
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Secondary reference #1
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Title
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Expression, Crystallization and preliminary x-Ray analysis of ligand-Free human glutathione s-Transferase m2-2.
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Authors
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L.N.Patskovska,
A.A.Fedorov,
Y.V.Patskovsky,
S.C.Almo,
I.Listowsky.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 1998,
54,
458-460.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1 (a) Diamond-shaped form A crystals under polarized
light. The size of the largest crystal in the photo is
approximately 0.3 × 0.2 × 0.2 mm. (b) Photograph of rod- and
needle-shaped form B crystals. The size of the central crystal
is approximately 0.4 × 0.2 × 0.2 mm.
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The above figure is
reproduced from the cited reference
with permission from the IUCr
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Secondary reference #2
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Title
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Crystal structure of human class mu glutathione transferase gstm2-2. Effects of lattice packing on conformational heterogeneity.
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Authors
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S.Raghunathan,
R.J.Chandross,
R.H.Kretsinger,
T.J.Allison,
C.J.Penington,
G.S.Rule.
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Ref.
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J Mol Biol, 1994,
238,
815-832.
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PubMed id
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Secondary reference #3
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Title
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Cloning, Expression, And characterization of a class-Mu glutathione transferase from human muscle, The product of the gst4 locus.
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Authors
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W.R.Vorachek,
W.R.Pearson,
G.S.Rule.
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Ref.
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Proc Natl Acad Sci U S A, 1991,
88,
4443-4447.
[DOI no: ]
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PubMed id
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