Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione.
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.
Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. johan.alander@ki.se
Johan
J
Alander
Johan
J
Lengqvist
Peter J
PJ
Holm
Richard
R
Svensson
Pascal
P
Gerbaux
Robert H H van den
RH
Heuvel
Hans
H
Hebert
William J
WJ
Griffiths
Richard N
RN
Armstrong
Ralf
R
Morgenstern
D000818:Animals; D001667:Binding, Competitive; D020134:Catalytic Domain; D004136:Dinitrobenzenes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008297:Male; D008861:Microsomes / Q000201:enzymology; D020836:Protein Structure, Quaternary; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization
D004136:Dinitrobenzenes; D004791:Enzyme Inhibitors; D008024:Ligands; C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase; D005978:Glutathione