Ia. Dubery et F. Smit, PHENYLALANINE AMMONIA-LYASE FROM COTTON (GOSSYPIUM-HIRSUTUM) HYPOCOTYLS - PROPERTIES OF THE ENZYME-INDUCED BY A VERTICILLIUM-DAHLIAE PHYTOTOXIN, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1207(1), 1994, pp. 24-30
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.