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 ⑀-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.

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113–119

“…Inhibition of PfGSTs by Hemin-Fresh hemin stock solutions were prepared as described previously (15). Inhibition of PfGSTs by hemin was studied by adding variable amounts of hemin (from 0.2 to 10 M) with a fixed enzyme concentration (1 M) in 1 ml of 0.1 M potassium phosphate buffer, pH 6.5, at 25°C.…”

“…The Integrity of Loop 113-119 Is Crucial for the Positive Cooperativity in Hemin Binding-PfGST displays homotropic behavior for hemin binding, so the affinity of the vacant subunit increases approximately 20 times when one hemin molecule binds to the first subunit of the dimer (15). This mechanism has been interpreted as a way to optimize the interception of hemin by the parasite that metabolizes large amounts of host cell hemoglobin and is continuously exposed to this toxic by-product.…”

“…An attempt to follow the reverse reaction, i.e. the dimer to tetramer transition after removal of GSH, was unsuccessful because of the very fast inactivation of this enzyme in the absence of GSH (15). Steady-state fluorescence anisotropy represents a useful approach for following a dimer/ tetramer transition because it provides a measure of the rotational motions of macromolecules (25).…”

“…Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13,16,17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13,18), provides insights into this effect.…”

“…Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13,16,17).…”