Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase

Almerinda Di Venere, Thomas Horn, Sabine Stehling, Giampiero Mei, Laura Masgrau, Àngels González-Lafont, Hartmut Kühn, Igor Ivanov

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10 Citations (Scopus)

Abstract

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme-substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages. © 2013 Elsevier B.V.
Original languageEnglish
Pages (from-to)1079-1088
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1831
Issue number6
DOIs
Publication statusPublished - 1 Jun 2013

Keywords

  • Fluorescence studies
  • Lipid peroxidation
  • Lipoxygenases
  • Molecular dynamics
  • Motional flexibility
  • Thermal stability

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