title: Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.
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.
authors.affiliation: Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Liebau
authors.forename: Francesca
authors.initials: F
authors.lastname: De Maria
authors.forename: Cora
authors.initials: C
authors.lastname: Burmeister
authors.forename: Markus
authors.initials: M
authors.lastname: Perbandt
authors.forename: Paola
authors.initials: P
authors.lastname: Turella
authors.forename: Giovanni
authors.initials: G
authors.lastname: Antonini
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Francesco
authors.initials: F
authors.lastname: Giansanti
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Stella
authors.forename: Mario
authors.initials: M
authors.lastname: Lo Bello
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Caccuri
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ricci
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019143:Evolution, Molecular; D005982:Glutathione Transferase / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008239:Lysine; D008956:Models, Chemical; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry; D010710:Phosphates / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology*; D017680:Potassium Compounds / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D001216:Asparagine; D006427:Hemin; C013216:potassium phosphate; D005982:Glutathione Transferase; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine; D009584:Nitrogen