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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

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IMPACT FACTOR 2017: 3.022

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1437-4315
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Volume 390, Issue 11

Issues

Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg257/Lys260, and unmasking of acid-base catalysis

Miriam Urscher
  • Butenandt Institute for Physiological Chemistry, Ludwig Maximilians University, D-81377 Munich, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Marcel Deponte
  • Butenandt Institute for Physiological Chemistry, Ludwig Maximilians University, D-81377 Munich, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2009-08-10 | DOI: https://doi.org/10.1515/BC.2009.127

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 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 Arg154 caused a 2.5-fold increase of K m app, 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 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 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.

Keywords: binuclear metallohydrolase; catalysis; glutathione; glyoxalase system; hydroxide formation; malaria

About the article

Corresponding author


Received: 2009-06-02

Accepted: 2009-07-16

Published Online: 2009-08-10

Published in Print: 2009-11-01


Citation Information: Biological Chemistry, Volume 390, Issue 11, Pages 1171–1183, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2009.127.

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