CHARACTERIZATION OF AUTHENTIC RECOMBINANT PEA-SEED LIPOXYGENASES WITHDISTINCT PROPERTIES AND REACTION-MECHANISMS

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pis um sativum L. cv. Birte) seeds have been cloned and expressed from ful l-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity f rom E, coli and pea seeds has confirmed the authenticity of the recomb inant products and established the properties of the native enzymes. D espite 86 % similarity at the amino acid sequence level, the enzymes h ave distinct properties. They have been characterized in terms of spec ific activity, Fe content, optimum pH, substrate and product specifici ty, apparent K-m and V-max, for the preferred substrate, linoleic acid , and interfacial behaviour with linoleic acid. We have used this evid ence, in addition to EPR spectroscopy of the hydroperoxide-activated e nzymes and estimates of k(cat)/K-m, to propose different reaction mech anisms for linoleic acid oxidation for the two isoforms. The differenc es relate primarily to carbonyl production from linoleic acid for whic h we propose a mechanism. This implicates the release of a peroxyl rad ical in an aerobic hydroperoxidase reaction, as the source of the carb onyl compounds formed by dismutation of the liberated peroxyl radical.