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

Abstract

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modu lated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113– 119 in the tetramerization process and in the cooperative phe nomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoen zymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tet ramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.