Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase.
The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.
Desideri
A
A
Department of Biology, University of Rome Tor Vergata, Italy.
Caccuri
A M
AM
Polizio
F
F
Bastoni
R
R
Federici
G
G
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011524:Protoporphyrins / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D008301:Maleimides; D011524:Protoporphyrins; D013438:Sulfhydryl Compounds; D006427:Hemin; C028025:protoporphyrin IX; D005982:Glutathione Transferase; D005978:Glutathione; C017514:S-methyl glutathione