Purification, product characterization and kinetic properties of soluble tomato lipoxygenase

Abstract

Soluble lipoxygenase (EC 1.13.11.12) from tomato fruits (Lycopersicon esculentum, var. Trust) was purified to apparent homogeneity as judged by SDS-PAGE, and the products and kinetics of the enzyme were studied in order to clarify the contradictory results that were obtained with a less purified enzyme. The specific activity of the enzyme, 668 nkat·mg−1 protein, was determined spectrophotometrically at pH 6.8 with a 150 mM linoleic acid solution containing 0.5 % methanol. Product analysis revealed that the enzyme mainly formed 9-hydroperoxy-(10E,12Z)-octadecadienoic acid (96 %) from linoleic acid with an enantiomeric excess of 82 % S. The 13-hydroperoxy-(9Z, 11E)-octadecadienoic acid formed (1 %) was racemic. Hydroxides and oxodienoic acids were negligible. Because these products were not observed under anaerobic conditions either, it was concluded that the pigment bleaching reported for tomato lipoxygenase does not result from release of intermediate free radicals to initiate the process of carbonyl formation. The influence of Tween-20 on the tomato enzyme was comparable to that of detergents on soybean lipoxygenase-1. A study of the variation of linoleic acid concentration between 0 and 20 mM in the absence of any detergent resulted in a typical Michaelis-Menten curve with a Km of 4.1 mM and a Vmax of 7.4 mM·min−1·mg−1 protein. The induction period of the progress curve of soluble tomato lipoxygenase was abolished by incubating the enzyme with 9-hydroperoxy-(10E,12Z)-octadecadienoic acid or with 13-hydroperoxy-(9Z, 11E)-octadecadienoic acid.