Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase

We have produced a model to define the linoleate-binding pocket of pea 9/13 -1ipoxygenase and have validated it by the construction and characterizatio n of eight point mutants. Three of the mutations reduced, to varying degree s, the catalytic centre activity (k(cat)) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and ma mmalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to di scuss the possible role of the mutated residues in pea 9/13-1ipoxygenase ca talysis. A new nomenclature for recombinant plant lipoxygenases based on po sitional specificity has subsequently been proposed. The null-effect of mut ating pea 9/13-lipoxygenase at the equivalent residue to that which control led dual positional specificity in cucumber 13/9-lipoxygenase, strongly sug gests that the mechanisms controlling dual positional specificity in pea 9/ 13-1ipoxygenase and cucumber 13/9-lipoxygenase are different. This was supp orted from modelling of another isoform of pea lipoxygenase, pea 13/9-lipox ygenase, Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linol eate and the volume of the linoleate-binding pocket rather than substrate o rientation. A single model for positional specificity, that has proved to b e inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxy genases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.