Abstract
The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr(50) in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr(50) to Ala. Although the key residue is located far from the catalytic center, the k(cat) value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k(cat) value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr(50), thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix alpha2, which interacts directly with GSH.
Highlights
Resistance of cells and organisms to electrophilic anticancer drugs, pesticides, and herbicides [5,6,7,8]
There are at least two ligand-binding sites per subunit: the glutathione-binding site (G-site), which is very specific for GSH and the hydrophobic substrate-binding site (H-site), which can bind a large variety of different electrophiles [18]
Amino acid residues in the dimer interface are not universally conserved in different GSTs classes, a particular lock and key motif is a common feature of the alpha, mu, and pi class enzymes (26 –28)
Summary
0.2 mM isopropyl-1-thio--D-galactopyranoside at 37 °C as described by Kolm et al [33]. After passing the mixture through glutathione-Sepharose 4B at 10 °C with a flow rate of 0.25 ml/min, the fractions containing GSTP1/Y50A were collected as wash (buffer A), while GSTP1-1 was collected as eluate (10 mM Tris-HCl, pH 7.8, containing 0.2 M NaCl, 5 mM S-hexylglutatione, 1 mM EDTA, 0.2 mM DTT, and 0.02%, w/v, NaN3). Inhibition Studies—The I50 values for each inhibitor for GSTP1-1, GSTP1/Y50A, and mutant Y50A were determined by measuring the specific activities at 30 °C in 0.1 M phosphate, pH 6.5, in the presence of 1 mM GSH, 1 mM CDNB, and different concentrations of inhibitor. Temperature Effect on the I50 Value—The I50 values for S-p-bromobenzyl glutathione for GSTP1-1, GSTP1/Y50A, and mutant Y50A were determined by measuring the specific activities at 20, 30, and 40 °C in 0.1 M phosphate, pH 6.5, in the presence of 0.33 mM GSH, 1 mM CDNB, and different concentrations of inhibitor. The enzymatic activity was measured in 0.1 M phosphate, pH 6.5, at 30 °C in the presence of 1 mM GSH and 1 mM CDNB
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