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Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione |
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| Authors: | Johan Å lander,Johan Lengqvist,Peter J. Holm,Pascal Gerbaux,Hans Hebert,Richard N. Armstrong |
| Affiliation: | a Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden b Karolinska Biomics Center, Karolinska University Hospital, SE-171 76 Stockholm, Sweden c Department of Biosciences, Karolinska Institutet, SE-141 57 Huddinge, Sweden d DMPK & Bioanalysis, Preclinical Development, Biovitrum AB, SE-112 76 Stockholm, Sweden e Laboratory of Organic Chemistry, Mass Spectrometry Center, University of Mons-Hainaut, 19 Avenue Maistriau, B-7000 Mons, Belgium f Utrecht University, Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands g Institute of Mass Spectrometry, School of Medicine, Room 352 Grove Building, Swansea University, Singleton Park, Swansea SA2 8PP, United Kingdom h Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA |
| Abstract: | The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (Kd = 320 ± 50 μM). All three product molecules could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, Kd could be determined for a low affinity GSH-binding site (2.5 ± 0.5 mM). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent. |
| Keywords: | GSH MGST1 MAPEG Alternating sites Cooperativity Glutathione transferase |
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