Investigation of the active site of human placenta glutathione transferase pi by means of a spin-labelled glutathione analogue.
A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.
Caccuri
A M
AM
Department of Biology, University of Rome Tor Vergata, Italy.
Polizio
F
F
Piemonte
F
F
Tagliatesta
P
P
Federici
G
G
Desideri
A
A
D001665:Binding Sites; D001667:Binding, Competitive; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D010920:Placenta / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D013113:Spin Labels; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine