Affinity labeling of the rabbit 12/15-lipoxygenase using azido derivatives of arachidonic acid

Biochemistry
Stepan RomanovIgor Ivanov

Abstract

Lipoxygenases are lipid-peroxidizing enzymes, which have been implicated in the pathogenesis of important diseases. They consist of a single polypeptide chain, which is folded into a two-domain structure. The large catalytic domain contains the putative substrate-binding pocket and the catalytic non-heme iron. To identify structural elements of the rabbit 12/15-lipoxygenase that are involved in enzyme/substrate and/or enzyme/product interaction, we synthesized a set of radioactively labeled lipoxygenase substrates carrying a photoreactive azido group (17-azido-ETE, 18-azido-ETE, 19-azido-ETE) and used these compounds as affinity probes. After photoaffinity labeling, the enzyme was digested proteolytically and modified tryptic cleavage peptides were identified by a combination of radio-HPLC and mass spectral analysis. Following this strategy, we observed covalent linkage of a cleavage peptide that contained Ile593, which has previously been identified as the sequence determinant for the positional specificity. These data are consistent with the previous suggestion that this peptide lines the substrate-binding pocket. Surprisingly, we also observed strong labeling of cleavage peptides originating from the N-terminal beta-barrel d...Continue Reading

References

Feb 1, 1996·Journal of Molecular Graphics·W HumphreyK Schulten
Jul 12, 2002·Journal of Molecular Modeling·Jason HemakThomas G Brock
May 5, 2004·The Journal of Biological Chemistry·Santosh NigamJesper Z Haeggström
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Citations

Oct 27, 2011·Biochemical Pharmacology·Joanna M WisniewskaBettina Hofmann
Aug 31, 2010·Archives of Biochemistry and Biophysics·Igor IvanovMatthias Walther
Jun 20, 2007·Trends in Biochemical Sciences·Olof RådmarkBengt Samuelsson
Feb 27, 2013·Biochimica Et Biophysica Acta·Almerinda Di VenereIgor Ivanov
May 1, 2010·The Journal of Physical Chemistry. B·Lea ToledoAngels González-Lafont

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