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

A series of chimeric human Mu class glutathione S-transferases were designe d to determine mechanisms by which they activate enzyme-bound glutathione ( GSH) for reaction with electrophilic substrates, In view of evidence that t he His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y . V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-120 2), 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 kinet ic properties invariably associated with enzymes containing an Arg107 resid ue include an inverse dependence of k(cat) on viscosity and lower K(m)GSH v alues relative to enzymes with other residues at that position. Moreover, a ffinities for GSH thiolate anion binding are greater for enzymes containing Arg(107), With K-d values of 20-50 mu M that are consistent with the K(m)G SH values (10-25 mu M) obtained by steady-state kinetic analyses. Both ther modynamic and kinetic and data indicate that the Arg(107) residue is specif ically involved in enhancing the binding affinity of GSH thiolate anion rel ative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that bot h Arg(107) and Tyr(6) residues are required for thiolate anion formation an d stabilization. The three-dimensional structure of ligand-free hGSTM2-2 de termined by x-ray crystallography suggests that Arg(107) maintains an elect rostatic interaction with the Asp(161) side chain (3 A apart), but is dista nt from the GSH-binding site. However, an alternative energetically favorab le model places the guanidino group 4 Angstrom from the sulfur atom of boun d GSH. It is suggested therefore, that in solution, motion of the positivel y charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the ob served greater affinity of enzymes containing Arg(107) for binding o f thiolate anion.