PDBsum entry 2gtu

Transferase

PDB id: 2gtu

Contents

Protein chain

217 a.a. *

Waters ×57

* Residue conservation analysis

PDB id:

2gtu

Name:

Transferase

Title:

Ligand-free human glutathione s-transferase m2-2 (E.C.2.5.1.18), monoclinic crystal form

Structure:

Glutathione s-transferase. Chain: a, b. Engineered: yes. Other_details: ligand-free

Source:

Homo sapiens. Human. Organism_taxid: 9606. Cell_line: hela. Cellular_location: cytoplasm. Gene: gstm2. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Other_details: the gstm2 cdna was amplified using rt-pcr and

Biol. unit:

Dimer (from PDB file)

Resolution:

2.55Å R-factor: 0.203 R-free: 0.257

Authors:

L.N.Patskovska,A.A.Fedorov,Y.V.Patskovsky,S.C.Almo,I.Listowsky

Key ref:

Y.V.Patskovsky et al. (2000). 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. J Biol Chem, 275, 3296-3304. PubMed id: 10652317 DOI: 10.1074/jbc.275.5.3296

Date:

26-May-98 Release date: 02-Mar-99

Protein chains

P28161  (GSTM2_HUMAN) -  Glutathione S-transferase Mu 2 from Homo sapiens

Seq: 218 a.a.

Struc: 217 a.a.

Enzyme reactions

• Enzyme class: E.C.2.5.1.18 - glutathione transferase.
• Reaction: RX + glutathione = an S-substituted glutathione + a halide anion + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.275.5.3296

J Biol Chem 275:3296-3304 (2000)

PubMed id: 10652317

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.

Y.V.Patskovsky, L.N.Patskovska, I.Listowsky.

ABSTRACT

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.

Selected figure(s)

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.

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) ( ).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 3296-3304) copyright 2000.

Figures were selected by an automated process.