Effect of the glutathione/glutathione disulfide redox couple on thiopurine methyltransferase.
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 concentrations and concentration ratios of the redox couple equaled those expected to occur in vivo. Activity measurements of the enzyme over time in these buffers at 30 degrees C indicated that thiol-disulfide exchange may be a part of the posttranslational modulation of hTPMT activity. Activity varied between 5% and 100%, with the lowest activities in buffers of low [GSH]/[GSSG] concentration ratios and of low total concentration of the redox couple. A thiol-disulfide exchange mechanism involving a mixed disulfide was proposed. Titration of the protein thiol groups with Ellmann's reagent (5,5'-dithiobis[2-nitrobenzoic acid]) revealed that at least two protein thiols were readily accessible for conjugation with the reagent, while others were conjugated more slowly. The previous model of hTPMT constructed by our group was in accordance with the experimental results. Inspection of the model indicated that one of the protein thiols subject to slow thiol-disulfide exchange may be situated at the binding site of the co-substrate of the enzyme and thus be responsible for the glutathione/glutathione disulfide modulation of the activity of hTPMT.
Lysaa
R A
RA
Department of Pharmacology, University of Tromso, Institute of Medical Biology, N-9038, Tromso, Norway. royal@fagmed.uit.no
Warren
D J
DJ
Sylte
I
I
Aarbakke
J
J
D002021:Buffers; D003198:Computer Simulation; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism*; D006801:Humans; D015122:Mercaptopurine / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis; D017186:Titrimetry
D002021:Buffers; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D005978:Glutathione; D019803:Glutathione Disulfide