Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis.
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 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.
Urscher
Miriam
M
Butenandt Institute for Physiological Chemistry, Ludwig Maximilians University, D-81377 Munich, Germany.
Deponte
Marcel
M
D001120:Arginine; D055162:Biocatalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology*; D012492:Salts / Q000494:pharmacology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000099301:Hydroxyacylglutathione Hydrolase
D001120:Arginine; D004791:Enzyme Inhibitors; D005978:Glutathione; D006878:Hydroxides; D008239:Lysine; D008670:Metals; D012492:Salts; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase; C031356:hydroxide ion