Purification and characterization of a phenylalanine ammonia-lyase from Ocimum basilicum
1996
Hao, Z. | Charles, D.J. | Yu, L. | Simon, J.E.
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 Mr 152000-153000; the intact subunit Mr was ca 38000. 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 Km and Vmax values of 329 micromolar and 11.43 micromol 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 Ki values of 19nM and 57 and 130 micromolar, respectively. Comparing the Ki 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.
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