Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg257/Lys260, and unmasking of acid-base catalysis
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Miriam Urscher
Abstract
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 kcat and pH-log kcat/Km profiles revealed acid-base catalysis. An acidic pKaapp 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 Arg154 caused a 2.5-fold increase of Kmapp, whereas replacements of Arg257 or Lys260 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 kcatapp 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 Arg257 and Lys260, 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.
©2009 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- Editorial
- Highlight: Molecular and Cellular Mechanisms of Memory
- Highlight: 60th Mosbach Colloquium of the GBM ‘Molecular and Cellular Mechanisms of Memory’
- Protein carboxyl methylation and the biochemistry of memory
- Chemotaxis: how bacteria use memory
- Mechanistic insights in light-induced cAMP production by photoactivated adenylyl cyclase alpha (PACα)
- Balance of power – dynamic regulation of chromatin in plant development
- Ultrafast memory loss and relaxation processes in hydrogen-bonded systems
- Memory and neural networks on the basis of color centers in solids
- Dissection of gene regulatory networks in embryonic stem cells by means of high-throughput sequencing
- The epigenetic bottleneck of neurodegenerative and psychiatric diseases
- Protein Structure and Function
- Mechanism of activation of Saccharomyces cerevisiae calcineurin by Mn2+
- Structural analysis of the choline-binding protein ChoX in a semi-closed and ligand-free conformation
- Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg257/Lys260, and unmasking of acid-base catalysis
- Genes and Nucleic Acids
- CA/C1 peptidases of the malaria parasites Plasmodium falciparum and P. berghei and their mammalian hosts – a bioinformatical analysis
- Proteolysis
- Placental expression of proteases and their inhibitors in patients with HELLP syndrome
- Irreversible inhibition of human cathepsins B, L, S and K by hypervalent tellurium compounds
