Cooperativity and Pseudo-cooperativity in the Glutathione S-Transferase from Plasmodium falciparum

Giorgio Federici,Lorenzo Stella,Cora Burmeister,Paola Turella,Eva Liebau,Francesca De Maria,Giorgio Ricci,Giovanni Antonini,Mario Lo Bello,Francesco Giansanti,Markus Perbandt,Anna Maria Caccuri

doi:10.1074/jbc.m503889200

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.

Highlights

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
A first indication for a cooperative interaction in glutathione S-transferases (GSTs) has been obtained for GSTP1-1, an isoenzyme that shows positive homotropic behavior by GSH binding after point mutation of crucial residues such as Cys-47 and Lys-54 [19, 20]
This phenomenon appears to be a particular case of a more general property of GSTP1-1 that uses cooperativity to oppose a complete loss of activity caused by physical or chemical killers [21]

Summary

EXPERIMENTAL PROCEDURES

Materials—GSH, 1-chloro-2,4-dinitrobenzene (CDNB), NBD-Cl, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F), and hemin were obtained from Sigma-Aldrich. Fluoride/Chloride Leaving Group Substitution—Kinetic data were obtained at 25 °C and at fixed NBD-Cl or NBD-F (0.2 mM) and GSH (1 mM) concentrations in 1 ml (final volume) of 0.1 M sodium acetate buffer, pH 5.0, containing suitable amounts of PfGST. Likewise, binding of hemin to PfGST was studied under the same experimental conditions by rapid mixing of 20 ␮M enzyme and 20 mM GSH in 0.1 M potassium phosphate buffer, pH 6.5, with the same volume of a hemin solution (from 10 to 100 ␮M). The affinity of PfGST (2 ␮M) for hemin was determined by measuring the perturbation of the intrinsic fluorescence of the protein following the addition of the inhibitor (from 0.5 to 5 ␮M) in the presence of 10 mM GSH in 0.1 M potassium phosphate buffer, pH 6.5. The MOLMOL program [30] was used for graphical interpretation and representation of results

RESULTS AND DISCUSSION

TABLE I Kinetic parameters for GSH binding to PfGST

TABLE II Kinetic parameters for hemin binding to PfGST