Affinity labeling of the rabbit 12/15-lipoxygenase using azido derivatives of arachidonic acid.
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 domain, and our mass spectral data suggested covalent linkage of oxidized affinity probes. Taken together, these results confirm the previous conclusion that Ile593 and surrounding amino acids are constituents of the active site, but they also implicate the N-terminal beta-barrel in enzyme/substrate and/or enzyme/product interaction.
Romanov
Stepan
S
Institute of Biochemistry, University Medicine Berlin-Charité, Monbijoustrasse 2, 10117 Berlin, Germany.
Wiesner
Rainer
R
Myagkova
Galina
G
Kuhn
Hartmut
H
Ivanov
Igor
I
D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D016718:Arachidonic Acid / Q000737:chemistry* / Q000378:metabolism; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors
C475064:12-15-lipoxygenase; C510173:18-azidoeicosa-5,8,11,14-tetraenoic acid; C510174:19-azidoeicosa-5,8,11,14-tetraenoic acid; D000345:Affinity Labels; D001095:Arachidonic Acids; D001386:Azides; D005227:Fatty Acids; D010455:Peptides; D016718:Arachidonic Acid; D001092:Arachidonate 12-Lipoxygenase; C458528:ALOX15B protein, human; D001093:Arachidonate 15-Lipoxygenase