Phenylalanine ammonia-lyase from cotton ( Gossypium hirsutum) hypocotyls: properties of the enzyme induced by a Verticillium dahliae phytotoxin

Phenylalanine ammonia-lyase (EC 4.3.1.5), induced by a Verticillium dahliae phytotoxin, has been purified to electrophoretic homogeneity from cotton hypocotyls by differential ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 52%. The enzyme is a tetramer with a molecular weight of 332000 to 337000. The isoelectric point is 4.6, and no isoforms were observed. The subunits of the enzyme are unstable and breaks down to fragments with M_r's of 69000 and 49500. The enzyme exhibited only activity with l-phenylalanine as substrate. Deamination was optimal at pH 8.9 and the activation energy was calculated as 100.6 kJ mol^-1. Non-Michaelian kinetics were observed with a K_m^L = 10.0 μM and K_m^H = 75.0 μM describing the binding of the substrate to the enzyme. Negative cooperative interactions occurred between the substrate binding sites with a Hill coefficient of 0.87. The inhibitors AOPP (S)-2-amino-oxy-3-phenylpropanoic acid), APEP (R)-1-amino-2-phenylethylphosphonic acid) and 2-AIP (2-aminoindan-2-phosphonic acid) strongly inactivated the enzyme, as did various analogues of l-phenylalanine and t-cinnamate. The induced enzyme is also sensitive to inhibition by phenylpropanoid intermediates and precursors involved in lignification such as 4-hydroxycinnamate and 3,4-dihydroxycinnamate.