Degradation of aromatic amino acids by fungi. II. Purification and properties of phenylalanine ammonia-lyase from Ustilago hordei.

Abstract

Phenylalanine ammonia-lyase (EC 4.3.1.5) isolated from the cells of Ustilago hordei was purified 128-fold. The purified enzyme catalyzed the stoichiometric deamination of L-phenylalanine to trans-cinnamic acid. The optimum pH was about 8.8 and the Michaelis constant for L-phenylalanine was 0.45 mM. The enzyme preparations also catalyzed the deamination of DL-m-fluorophenylalanine, DL-p-fluorophenylalanine, and DL-m-hydroxyphenylalanine, although there was greater affinity of L-phenylalanine. The enzymic product from m-hydroxyphenylalanine was isolated and characterized as m-coumaric acid. No deamination of tyrosine could be demonstrated either with crude or purified preparations. Apparently there was no cofactor or metal ion requirement for the reaction. Sulfhydryl inhibitors like p-chloromercuribenzoate and N-ethylmaleimide partially inhibited the reaction in the presence of ethylenediaminetetracetate. The high susceptibility of the enzyme to inactivation by heavy metal ions like Ag+, Hg+, and Cd++also suggest the involvement of sulfhydryl groups in the reaction. Several compounds structurally related to phenylalanine were inhibitory, trans-Cinnamic acid exerted strong product inhibition. Reversibility of the reaction was demonstrated.