Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Abstract

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG ؉ NADPH ؉ H ؉ 7 2 GSH ؉ NADP ؉ ). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative halfreaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E ox -EH 2 and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K eq from the Haldane relationship, 0.075, very similar to the K eq of the same reaction for the yeast enzyme ( (S-S)⅐NADP؉ and E(FAD)(SH) 2 ⅐NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.The glutathione disulfide/glutathione (GSSG/GSH) redox couple is present in most eukaryotic cells at millimolar concentrations (1). Major functions of GSH include the detoxification of reactive oxygen species by donation of reducing equivalents to glutathione peroxidase and glutathione S-transferase as well as enzyme regulation by thiol-disulfide interchange reactions (2). The flavoenzyme glutathione reductase (GR, 1 EC 1.6.4.2) catalyzes the reduction of GSSG to GSH and creates an intracellular GSH/GSSG ratio of 20 to 1000 depending on the respective metabolic conditions (3). Thus, glutathione reductase, a protein of 2 ϫ 55 kDa, plays a key role in cellular redox homeostasis. Both human GR (hGR) and Plasmodium falciparum GR (PfGR) are essential for the survival of the malarial parasite within the human erythrocyte (4, 5). The amino acid sequence identity between the two enzymes is 45% with two major features distinguishing them: (i) an insertion of 34 amino acids within the central domain (residues 318 -351) between the FAD binding motif and the highly conserved H8 helix (6); (ii) the differences in the amino acid residues lining the wall of the interface cavity, where only 8 out of 25 residues are conserved in PfGR. This cavity represents the binding site of many inhibitors including isoalloxazines, safranine, and menadione in human GR and could be the binding site of methylene blue in PfGR (7).Glutathione reductase belongs to the pyridine nucleotidedisulfide oxidoreductase family of homodimeric flavoenzymes that includes lipoamide dehydrogenase and thioredoxin reductase. Like other members of this family, each subunit of GR contains a disulfide and a flavin that are in redox contact. This ground state of the enzyme is ...

The Mechanism of High M r Thioredoxin Reductase from Drosophila melanogaster

“…1A) (4,12,16,18). Reduction of DmTrxR-1 was detected as a decrease in the flavin absorbance maximum (⑀ 462 nm ϭ 11.9 mM Ϫ1 cm Ϫ1 for E ox ), and this was accompanied by an increase in absorbance around 540 nm that was caused by a charge-transfer interaction between the thiolate of Cys 62 and the isoalloxazine ring.…”

“…All reagents and other enzymes were obtained from Serva and Sigma and were of highest available purity. Titrations and stopped-flow experiments were conducted under anaerobic conditions using methods described previously (18), and all reactions were conducted in 100 mM potassium phosphate buffer, pH 7.0.…”

Interactions of Quinones with Thioredoxin Reductase

“…According to the Haldane relationship, the ratio of the bimolecular rate constants of forward and reverse reactions gives the equilibrium constant of the reaction (K), which in turn is related to the difference in the standard redox potential of the reactants (⌬E 0 7 ϭ 29.5 mV ϫ log K for a two-electron transfer). This relation, based on the rates of enzyme reactions with NADP(H) (E 0 7 ϭ Ϫ0.320 V), has been used for calculating E 0 7 of yeast (39) and P. falciparum glutathione reductases (40). For Reaction 1, the ratio of k cat /K m for NADPH oxidation and NADP ϩ reduction (Table I) gives K ϭ 8.07 Ϯ 0.5, and E We next analyzed the effect of NADP ϩ on the NADPH-dependent reduction of DTNB by TrxR.…”

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

“…According to the literature, it is an EH 4 form of a given disulfide reductase that reduces nonphysiological substrates such as quinones and O 2 as a "diaphorase" (4,7). This EH 4 form is readily accessible in LipDH and in P. falciparum GR but not in human GR (5,15,56).…”