title: Phenylalanine ammonia-lyase from cotton (Gossypium hirsutum) hypocotyls: properties of the enzyme induced by a Verticillium dahliae phytotoxin.
abstract: 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 332,000 to 337,000. 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 69,000 and 49,500. 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 KmL = 10.0 microM and KmH = 75.0 microM 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.
authors.affiliation: Department of Chemistry and Biochemistry, RAU-University, South Africa.
authors.forename: I A
authors.initials: IA
authors.lastname: Dubery
authors.forename: F
authors.initials: F
authors.lastname: Smit
mesh_terms: D004790:Enzyme Induction / Q000187:drug effects; D003368:Gossypium / Q000201:enzymology*; D003904:Mitosporic Fungi / Q000737:chemistry; D010650:Phenylalanine Ammonia-Lyase / Q000096:biosynthesis*; D010940:Plant Proteins / Q000096:biosynthesis*; D014118:Toxins, Biological / Q000494:pharmacology
chemical_list: D010940:Plant Proteins; D014118:Toxins, Biological; D010650:Phenylalanine Ammonia-Lyase