No abstract

SummaryThe ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer-dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methylgerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum.

Glyoxalase II (GloII) is a ubiquitous thioester hydrolase catalyzing the last step of the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Here, we present a detailed structure-function analysis of cGloII from the malaria parasite Plasmodium falciparum. The activity of the enzyme was salt-sensitive and pH-log k cat and pH-log k cat /K m profiles revealed acidbase catalysis. An acidic pK a app value of approximately 6 probably reflects hydroxide formation at the metal center. The glutathione-binding site was analyzed by site-directed mutagenesis. Substitution of residue Arg 154 caused a 2.5-fold increase of K m app , whereas replacements of Arg 257 or Lys 260 were far more detrimental. Although the glutathione-binding site and the catalytic center are separated, six of six single mutations at the substrate-binding site decreased the k cat app value. Furthermore, product inhibition studies support a Theorell-Chance Bi Bi mechanism with glutathione as the second product. We conclude that the substrate is predominantly bound via ionic interactions with the conserved residues Arg 257 and Lys 260, and that correct substrate binding is a pH-and salt-dependent rate-limiting step for catalysis. The presented mechanistic model is presumably also valid for GloII from many other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases.

Glucose consumption and therefore methylglyoxal production of human erythrocytes increase significantly upon infection with malaria parasites. The glyoxalase systems of the host–parasite unit cope with this metabolic challenge by catalyzing the removal of harmful methylglyoxal. Thus, glyoxalase 1 from the malaria parasite Plasmodium falciparum (PfGlo1) could be a promising drug target. However, the enzyme has two different active sites and their simultaneous inactivation is considered challenging. Here, we describe the inactivation of PfGlo1 by two glyoxalase‐specific tight‐binding inhibitors with nanomolar Kiapp values and noncompetitive inhibition patterns. The inhibitors do not discriminate between the high‐affinity and the high‐activity conformations of PfGlo1, but seem to stabilize or trigger a conformational change in analogy with the substrate. In summary, we have characterized the most potent inhibitors of PfGlo1 known to date.