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Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione
- Publication Year :
- 2009
-
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 (K(d)=320+/-50 microM). 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, K(d) 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.
- Subjects :
- Male
Spectrometry, Mass, Electrospray Ionization
Biophysics
Trimer
Cooperativity
In Vitro Techniques
Ligands
Binding, Competitive
Biochemistry
Article
Rats, Sprague-Dawley
chemistry.chemical_compound
Catalytic Domain
Microsomes
Animals
Reactivity (chemistry)
Enzyme Inhibitors
Protein Structure, Quaternary
Molecular Biology
Glutathione Transferase
biology
Ligand
Active site
Glutathione
Rats
Dinitrobenzenes
Kinetics
chemistry
biology.protein
Microsome
Peroxidase
Subjects
Details
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....4b62b94be2842a8d3ef3b82750e31749