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.
Alander
Johan
J
Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. johan.alander@ki.se
Lengqvist
Johan
J
Holm
Peter J
PJ
Svensson
Richard
R
Gerbaux
Pascal
P
Heuvel
Robert H H van den
RH
Hebert
Hans
H
Griffiths
William J
WJ
Armstrong
Richard N
RN
Morgenstern
Ralf
R
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