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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 acid-base catalysis. An acidic p K  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.

biological chemistry

Plasmodium falciparum  glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg 257 /Lys 260 , and unmasking of acid-base catalysis