Purification and Characterization of Phenylalanine Ammonia Lyase from Trichosporon cutaneum
Andrea Goldson-Barnaby1, Christine H Scaman
1Food, Nutrition, and Health, University of British Columbia, 2205 East Mall, Vancouver, BC, Canada V6T 1Z4 ; Department of Chemistry, University of the West Indies, Kingston, Jamaica.
Abstract:
Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (1.6 ± 0.3 : 0.4 ± 0.1 μ mol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8-8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, K m 5.0 ± 1.1 mM) and positive allostery (Tyr, K' 2.4 ± 0.6 mM, Hill coefficient 1.9 ± 0.5) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme's ability to act on both substrates.
