Purification and characterization of a phenylalanine ammonia-lyase from Ocimum basilicum

A new phenylalanine ammonia-lyase (PAL) was purified from leaves of Ocimum basilicum L. (chemotype, methyl cinnamate). Separation techniques applied included anion exchange chromatography and preparative electroelution from a non-denaturing polyacrylamide gel. A 180-fold purification was obtained. The native enzyme was a homotetramer of M(r) 152 000-153 000; the intact subunit M(r) was ca 38 000. The enzyme catalysed the conversion of L- phenylalanine-d₈ into trans-cinnamic acid-d₇, as determined by GC-mass spectral analysis of silylated reaction products. The purified native enzyme had K(m) and V(max) values of 329 μM and 11.43 μmol min^-1 mg^-1 protein, respectively, for L-phenylalanine and was competitively inhibited by 2- amino-indan-2-phosphonic acid, trans-cinnamic acid and trans-methyl cinnamate with K(i) values of 19 nM and 57 and 130 μM, respectively. Comparing the K values between trans-cinnamic acid and trans-methyl cinnamate for L- phenylalanine indicated that the regulation of PAL is not only related to the mechanism of feedback inhibition in the biosynthesis of trans-methyl cinnamate.