Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Liebau, E., De Maria, F., Burmeister, C., Perbandt, M., Turella, P., Antonini, G., et al. (2005). Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 280(28), 26121-26128 [10.1074/jbc.M503889200].

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum

FEDERICI, GIORGIO;STELLA, LORENZO;LO BELLO, MARIO;CACCURI, ANNA MARIA;RICCI, GIORGIO
2005-07-15

Abstract

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.
15-lug-2005
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
Settore BIO/12 - BIOCHIMICA CLINICA E BIOLOGIA MOLECOLARE CLINICA
Settore CHIM/02 - CHIMICA FISICA
English
Con Impact Factor ISI
Animals; Spectrometry, Fluorescence; 4-Chloro-7-nitrobenzofurazan; Glutathione Transferase; Lysine; Hemin; Asparagine; Potassium Compounds; Inhibitory Concentration 50; Protein Conformation; Models, Molecular; Recombinant Proteins; Dimerization; Enzyme Inhibitors; Plasmodium falciparum; Protein Binding; Evolution, Molecular; Binding Sites; Static Electricity; Phosphates; Sulfhydryl Compounds; Kinetics; Models, Chemical; Catalysis; Nitrogen
Liebau, E., De Maria, F., Burmeister, C., Perbandt, M., Turella, P., Antonini, G., et al. (2005). Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 280(28), 26121-26128 [10.1074/jbc.M503889200].
Liebau, E; De Maria, F; Burmeister, C; Perbandt, M; Turella, P; Antonini, G; Federici, G; Giansanti, F; Stella, L; LO BELLO, M; Caccuri, Am; Ricci, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/37199
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