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 da hliae phytotoxin, has been purified to electrophoretic homogeneity fro m cotton hypocotyls by differential ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 52%. The enzy me is a tetramer with a molecular weight of 332 000 to 337 000. The is oelectric 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 6 9 000 and 49 500. The enzyme exhibited only activity with L-phenylalan ine as substrate. Deamination was optimal at pH 8.9 and the activation energy was calculated as 100.6 kJ mol(-1). Non-Michaelian kinetics we re observed with a K-m(L) = 10.0 mu M and K-m(H) = 75.0 mu M describin g the binding of the substrate to the enzyme. Negative cooperative int eractions occurred between the substrate binding sites with a Hill coe fficient of 0.87. The inhibitors AOPP (S)-2-amino-oxy-3-phenylpropanoi c acid), APEP (R)-1-amino-2-phenylethylphosphonic acid) and 2-AIP (2-a minoindan-2-phosphonic acid) strongly inactivated the enzyme, as did v arious analogues of L-phenylalanine and t-cinnamate. The induced enzym e is also sensitive to inhibition by phenylpropanoid intermediates and precursors involved in lignification such as 4-hydroxycinnamate and 3 ,4-dihydroxycinnamate.