Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis. - Related Articles
Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis.
Biol Chem. 2009 Aug 10;
Authors: Urscher M, Deponte M
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 enzymatic activity of cGloII was salt-sensitive and pH-log k(cat) and pH-log k(cat)/K(m) profiles revealed acid-base catalysis with a rather constant pK(a)(app) of ~6 and a variable basic pK(a)(app) value. The acidic pK(a)(app) probably reflects hydroxide formation at the metal center. The role of the glutathione-binding site for catalysis 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 and increased K(m)(app) about 20- to 50-fold. Although the glutathione-binding site and the water-activating catalytic center are separated, six of six single mutations at the substrate-binding site had a significant negative influence on k(cat)(app). In addition, 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. Due to the structural conservation of the enzyme, the presented mechanistic model is presumably also valid for GloII from other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases.
PMID: 19663684 [PubMed - as supplied by publisher] [PubMed-Malaria]