Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms.

摘要: The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned expressed full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison both purified to apparent homogeneity E. coli has confirmed the authenticity recombinant products established properties native enzymes. Despite 86% similarity at amino acid sequence level, enzymes distinct properties. They characterized terms specific activity, Fe content, optimum pH, substrate product specificity, Km Vmax for preferred substrate, linoleic acid, interfacial behaviour with acid. We used this evidence, addition EPR spectroscopy hydroperoxide-activated estimates kcat/Km, propose different reaction mechanisms oxidation isoforms. differences relate primarily carbonyl production which we a mechanism. This implicates release peroxyl radical an aerobic hydroperoxidase reaction, source compounds formed by dismutation liberated radical.