Plasmodium falciparum glutathione S-transferase--structural and mechanistic studies on ligand binding and enzyme inhibition

Abstract

Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.

Figure 1.

Structural comparison of GST enzymes. Superposition of the enzyme–inhibitor structures of PfGST (yellow), mu-class human GST (red, 1c72), and pi-class human GST (blue, 11gs). All inhibitors are drawn in ball and stick, with oxygens red and nitrogens blue. PfGST with bound S-hexylglutathione and a polyethylene glycol (PEG) molecule (carbons: yellow). Human mu-class GST with a bound glutathione conjugate (1-hydroxy-2-S-glutathionyl-3-paranitrophenoxypropane) (carbons: red). Human pi-class GST with the bound glutathione conjugate of ethacrynic acid (carbons: blue). The figure was drawn using the programs molscript and raster3d (Kraulis 1991; Merritt and Bacon 1997).

Figure 2.

Equivalent views of the H-site. Close-up of native and liganded PfGST (A, B, gold) and a pi-GST (C, brown, 11gs), focusing on the active site. The active site is occupied by glutathione conjugate moieties (green, B, C). In PfGST, H-site residues interact with a PEG molecule (blue, B) or with the loop of monomer B (blue, A), which protrudes into monomer A resulting in a marked change in the position of Phe 116. Mutated residues are shown in black. The figure was drawn using the programs molscript and raster3d (Kraulis 1991; Merritt and Bacon 1997).

Figure 3.

Inhibition of PfGST by ferriprotoporphyrin IX. The parallel lines in a Dixon plot indicate an uncompetitive inhibition type of PfGST by FP. The corresponding Cornish-Bowden diagram ([S]/v against [I]) yields a K_i value of 3 μM (data not shown).

Figure 4.

Binding of ferriprotoporphyrin IX to wild-type and mutant PfGST monitored by fluorescence quenching. Quenching of the intrinsic fluorescence of wild-type and mutant PfGST following successive additions of FP in the presence of 1 mM GSH. All titrations have been reproduced at least three times yielding values which differed by <3%.