Partial purification and some properties of phenylalanine ammonia-lyase of tobacco (Nicotiana tabacum).

The enzyme responsible for the first step in the synthesis of phenolic compounds in tobacco, L-phenylalanine ammonia-lyase, was purified approximately 200-fold from leaves of Nicotiana tabacum var. Burley 21. The purest preparation was at least 70 per cent homogenous as defined by disc electrophoresis. The optimal pH for the reaction was between 8·0 and 8·6. At pH 7·4, the Km for the substrate L-phenylalanine was 1·6 × 10?4 M and at pH 8·55 it was 2·2 × 10?4 M. D-Phenylalanine, L-tyrosine, m-tyrosine, o-tyrosine, and dihydroxyphenylalanine were not deaminated by the enzyme. Sulfhydryl compounds caused a significant stimulation of activity, but no cofactor requirements were found. The enzyme was sharply inhibited by trans-cinnamic acid, in a competitive manner, with a Ki of 3·1 × 10?5 M at pH 7·1. Other phenolic inhibitors included o-coumaric acid, o-tyrosine, and quercetin. It is suggested that trans-cinnamic acid may play a role in vivo in regulating biosynthesis of phenolic compounds in tobacco.