the glutathionebinding site in glutathione stransferases investigation of the cysteinyl glycyl and gammaglutamyl domains the gshbinding site of glutathione stransferase gst isoenzymes was studied by investigating their substratespecificity for three series of gsh analogues further a model of the interactions of gsh with the gsite is proposed twelve glycylmodified gsh analogues four ester derivatives of gsh and three cysteinylmodified gsh analogues were synthesized and tested with purified forms of rat liver gst 11 22 33 and 44 the glycyl analogues exhibited spontaneous chemical reaction rates with 1chloro24dinitrobenzene comparable with the gsh rate in contrast the enzymic rates vmax differed greatly from less than 1 up to 140 mumolmin 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 vmax values for the analogues analogues of gsh in which lcysteine was replaced by dcysteine lhomocysteine or lpenicillamine showed little or no capacity to replace gsh as cosubstrate for the gsts gsh monomethyl and monoethyl esters showed vmax values greater than the vmax measured with gsh the vmax for the monoethyl ester of gsh and gst 33 was 5fold that for gsh the data obtained in this and previous studies adang brussee meyer coles ketterer van der gen mulder 1988 biochem j 255 721724 adang meyer brussee van der gen ketterer mulder 1989 biochem j 264 759764 allow a model of the interactions of gsh in the gsite in gsts to be postulated the gammaglutamyl site is the main binding determinant the alphacarboxylate group is obligatory whereas shifting of the amino group and shortening of the peptide backbone only decreased kcatkm 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 gsite of gsts amino acid analogues all showed km values between 02 and 06 mm that for gsh is 0203 mm but large differences in vmax 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 vmax in some analogues are briefly discussed