Chemical Modification of 5-Lipoxygenase from the Korean Red Potato

다국어 초록 (Multilingual Abstract)

The lipoxygenase was purified 35fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis and Sepharose 6B column chromatography. The purified enzyme with 2 M (NH₄)₂SO₄ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at-20。C. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenases purified from the red potato were found to be pH 9.0. and 30。C, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were 48μM and 0.03 μM per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10 mM EDTA, and 1 mM NaN₃), but was inhibited by several divalent cations, such as Cu??, Co?? and Ni??. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward’s reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) proceeded in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.