Effect of the glutathione/glutathione disulfide redox couple on thiopurinemethyltransferase

The susceptibility of recombinant human thiopurine methyltransferase (hTPMT ) to thiol-disulfide exchange was investigated. The enzyme was incubated in buffers of the redox couple GSH and GSSG. The values of the chosen concent rations and concentration ratios of the redox couple equaled those expected to occur in vivo. Activity measurements of the enzyme over time in these b uffers at 30 degreesC indicated that thiol-disulfide exchange may be a part of the posttranslational modulation of hTPMT activity. Activity varied bet ween 5% and 100%, with the lowest activities in buffers of low [GSH]/[GSSG] concentration ratios arid of low total concentration of the redox couple. A thiol-disulfide exchange mechanism involving a mixed disulfide was propos ed. Titration of the protein thiol groups with Ellmann's reagent (5,5'-dith iobis[2-nitrobenzoic acid]) revealed that at least two protein thiols were readily accessible for conjugation with the reagent, while others were conj ugated more slowly. The previous model of hTPMT constructed by our group wa s in accordance with the experimental results. Inspection of the model indi cated that one of the protein thiols subject to 'slow thiol-disulfide excha nge may be situated at the binding site of the co-substrate of the enzyme a nd thus be responsible for the glutathione/glutathione disulfide modulation of the activity of hTPMT. (C) 2001 Elsevier Science Inc. All rights reserv ed.