THE GLUTATHIONE-BINDING SITE IN GLUTATHIONE S-TRANSFERASES - INVESTIGATION OF THE CYSTEINYL, GLYCYL AND GAMMA-GLUTAMYL DOMAINS

The GSH-binding site of glutathione S-transferase (GST) isoenzymes was studied by investigating their substrate-specificity for three series of GSH analogues; further, a model of the interactions of GSH with the G-site is proposed. Twelve glycyl-modified GSH analogues, four ester derivatives of GSH and three cysteinyl-modified GSH analogues were synthesized and tested with purified forms of rat liver GST (1-1, 2-2, 3-3 and 4-4). The glycyl analogues exhibited spontaneous chemical reaction rates with 1-chloro-2,4-dinitrobenzene comparable with the GSH rate. In contrast, the enzymic rates (V(max.)) differed greatly, from less than 1 up to 140 μmol/min per mg; apparently, a reaction mechanism is followed that is very sensitive to substitutions at the glycyl domain. No correlation exists between the chemical rates and V(max.) values for the analogues. Analogues of GSH in which L-cysteine was replaced by D-cysteine, L-homocysteine or L-penicillamine showed little or no capacity to replace GSH as co-substrate for the GSTs. GSH monomethyl and monoethyl esters showed V(max.) values greater than the V(max.) measured with GSH: the V(max.) for the monoethyl ester of GSH and GST 3-3 was 5-fold that for GSH. The data obtained in this and previous studies [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen and Mulder (1988) Biochem. J. 255, 721-724; Adang, Meyer, Brussee, van der Gen, Ketterer and Mulder (1989) Biochem. J. 264, 759-764] allow a model of the interactions of GSH in the G-site in GSTs to be postulated. The γ-glutamyl site is the main binding determinant: the α-carboxylate group is obligatory, whereas shifting of the amino group and shortening of the peptide backbone only decreased k(cat.)/K(m). Furthermore, the GSTs appear to be very critical with respect to a correct orientation of the thiol group of the GSH analogue. The glycyl site is the least restrictive domain in the G-site of GSTs: amino acid analogues all showed K(m) values between 0.2 and 0.6 mM (that for GSH is 0.2-0.3 mM), but large differences in V(max.) exist. The glycyl carboxylate group is not essential for substrate recognition, since decarboxy analogues and ester derivatives showed high activities. The possible mechanisms for an increased V(max.) in some analogues are briefly discussed.