Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue.
Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008623:Mercaptoethanol / Q000031:analogs & derivatives*; D051381:Rats; D011919:Rats, Inbred Strains; D013113:Spin Labels / Q000138:chemical synthesis*
D000596:Amino Acids; D003497:Cyclic N-Oxides; D013113:Spin Labels; C030190:S-(2,4-dinitrophenyl)glutathione; D008623:Mercaptoethanol; C040264:SL-glutathione; C040255:SL-mercaptoethanol; D005982:Glutathione Transferase; D005978:Glutathione