were determined using 1 chloro 2 4 dinitrobenzene as substrate mannervik b danielson u h crc critical reviews in biochemistry 1988 23 283 337 the reaction rate profile conformed to a double exponential decay composed of a rapid enzyme dependent initial phase involving 1b followed by a slower enzyme independent first order phase fig2 traces b and c this finding was consistent with the mechanism shown in scheme 4 whereby an initial enzyme catalyzed michael addition of gsh to 1b afforded the exocyclic enone 3 once dissociated from the enzyme free 3 reacted with gsh nonenzymatically to form 2b when 1b 0 1 mm was incubated with cysteine 0 5 mm and gsh 0 5 mm in the presence of gstp 1 1 1 5 units for 30 min and the reaction mixture fractionated by reverse phase hplc thiol adducts 2b and 4 were isolated based on the integrated intensities of the well resolved peaks corresponding to 4 u02dc 9 5 min and 2b u02dc 1 5 min the product ratio in the presence of gstp1 1 was identical to that obtained from a nonenzymatic incubation of 1b with the same concentrations of cysteine and gsh fig3 that finding indicated that 3 dissociated from the enzyme prior to reacting with the free thiols in solution and was consistent with the mechanism in scheme 4 adduct 2b 0 5 mm was stable under the reaction conditions undergoing less than 1 conversion to 4 in the.