INVOLVEMENT OF THE CARBOXYL GROUPS OF GLUTATHIONE IN THE CATALYTIC MECHANISM OF HUMAN GLUTATHIONE TRANSFERASE A1-1

The present study proposes the participation of both carboxylate group s of the glutathione molecule as functional entities in the catalytic apparatus of human glutathione transferase (GST) A1-1. Functional stud ies in combination with structural data provide evidence for the alpha -carboxylate of the Glu residue of glutathione acting as a proton acce ptor in the catalytic mechanism. The Glu alpha-carboxylate is hydrogen -bonded to a protein hydroxyl group and a main-chain NH, as well as to a water molecule of low mobility in the active site region. The Glu o r-carboxylate of glutathione is bound in a similar manner to the activ e sites of mammalian glutathione transferases of classes Alpha, Mu, an d Pi, for which three-dimensional structures are known. Mutation of th e hydroxyl group that is hydrogen-bonded to the alpha-carboxylate of t he Glu residue of glutathione (Thr68-->Val) caused a shift of the pH d ependence of the enzyme-catalyzed reaction, suggesting that the acidic Limb of the pH-activity profile reflects the ionization of the carbox ylate of the Glu residue of glutathione. The second carboxylate group of glutathione, which is part of its Gly residue, interacts with two A rg side chains in GST A1-1. One of these residues (Arg45) may influenc e an ionic interaction (Arg221/Asp42), which appears to contribute to binding of the second substrate by fixing the C-terminal alpha-helix a s a lid over the active site. Removal of the Gly residue from the glut athione molecule caused a 13-fold increase in the K-M value for the el ectrophilic substrate. Thus, the Gly carboxylate of glutathione, by wa y of influencing the topology of the active site, contributes to the b inding of the second substrate of the enzyme. Consequently, the glutat hione molecule has several functions in the glutathione transferase ca talyzed reactions, not only as a substrate providing the thiol group f or different types of chemical reactions but also as a substrate contr ibuting a carboxylate that acts as a proton acceptor in the catalytic mechanism and a carboxylate that modulates binding of the second subst rate to the enzyme.