Calorimetric Studies of Ligands Binding to Glutathione S-Transferase from the Malarial Parasite Plasmodium falciparum

Abstract

Glutathione S-transferase, from the malarial parasite Plasmodium falciparum (PfGST), exerts a protective role in the organism and is thus considered an interesting target for antimalarial drug development. In contrast to other GSTs, it is present in solution as a tetramer and a dimer in equilibrium, which is induced by glutathione (GSH). These properties prevent a calorimetric titration from being conducted upon binding of ligands to this protein's G-site. Thermodynamic characterization can be an optimal strategy for antimalarial drug development, and isothermal titration calorimetry (ITC) is the only technique that allows the separation of the binding energy into both enthalpic and entropic contributions. This information facilitates an understanding of the changes in the drugs' substituents, improving their affinity and specificity. In this study, we have applied a nontypical ITC procedure, based on the dissociation of the ligand-protein complex, to calorimetrically study the binding of the GSH substrate, and the glutathione sulfonate competitive inhibitor, to dimeric PfGST over a temperature range of 15-37 °C. The optimal experimental conditions for applying this procedure have been optimized by studying the dimer to tetramer conversion using size exclusion chromatography. The binding of these ligands to dimeric PfGST is noncooperative, the affinity of glutathione sulfonate being approximately 2 orders of magnitude higher than that of its natural substrate GSH. The binding of both ligands is enthalpically favorable and entropically unfavorable at all the studied temperatures. These results demonstrate that, although PfGST presents differences when compared to other known GSTs, these ligands bind to its dimeric form with a similar affinity and energetic balance. However, in contrast to that of other GSTs, the binding of GSH to protein, in the absence of the ligand, is slow.