| Abstract
| - Lipoxygenases are lipid-peroxidizing enzymes, which have been implicated in the pathogenesisof important diseases. They consist of a single polypeptide chain, which is folded into a two-domainstructure. 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 lipoxygenasesubstrates carrying a photoreactive azido group (17-azido-ETE, 18-azido-ETE, 19-azido-ETE) and usedthese compounds as affinity probes. After photoaffinity labeling, the enzyme was digested proteolyticallyand modified tryptic cleavage peptides were identified by a combination of radio-HPLC and mass spectralanalysis. 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. Thesedata 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 β-barreldomain, 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 ofthe active site, but they also implicate the N-terminal β-barrel in enzyme/substrate and/or enzyme/productinteraction.
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