KINETICS, REGULATION AND PURIFICATION OF L-PHENYLALANINE AMMONIA-LYASE FROM GERMINATING LETTUCE SEEDS (PHENYLPROPANOIDS, ENZYME, ALPHA-AMINOXY - BETA-PHENYLPROPIONIC ACID)

<p>The kinetics of L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in germinating lettuce (Lactuca sativa cv. Grand Rapids) seeds was investigated. The Km of the enzyme was determined to be 4.2 x 10('-5) M. PAL in lettuce seeds did not show tyrosine ammonia-lyase activity. The nature of inhibition of PAL by various substrate analogues and phenylpropanoid compounds was studied. Substrate analogues like D-phenylalanine, p-fluorophenylalanine, (beta)-phenyllactic acid and tryptophan were found to inhibit PAL competitively whereas tyrosine did not show any inhibition of enzyme activity. Of the phenylpropanoids used, cinnamic acid was found to be a competitive inhibitor whereas chlorogenic acid showed mixed inhibition. Other phenylpropanoid compounds like p-coumaric acid, caffeic acid, coumarin, quercetin, ferulic acid etc. did not show any inhibition of PAL activity in vitro.</p><p>The regulation of PAL activity by various substrate analogues, intermediates and endproducts of the phenylpropanoid pathway in relation to the growth of the embryonic axes was studied. The substrate, L-phenylalanine, its D-isomer and (beta)-phenyllactic acid did not show any significant effect on PAL activity at low concentrations (50 and 100 uM), whereas at higher concentrations (500 uM) L-phenylalanine inhibited both PAL activity and radicle elongation. On the other hand, D-phenylalanine promoted both. Other substrate analogues like 2-aminoxy-3-phenylpropionic acid (AOPP), p-fluorophenylalanine and tryptophan showed a strong inhibition of PAL activity, the inhibition being concentration dependent. Another substrate analogue, tyrosine, stimulated both PAL activity and radicle elongation.</p><p>The phenylpropanoid compounds were inhibitors of PAL activity at higher concentrations. At lower concentrations, there was a concentration dependent inhibition of PAL activity and radicle elongation. In all these treatments, a strong correlation was observed between PAL activity and radicle length.</p><p>PAL was purified 37 fold from 40 h old lettuce seedlings. Excised radicles were used as a source of the enzyme for purification since more than 95% of the PAL activity was localized in the radicles. A combination of four techniques viz. ammonium sulfate precipitation, gel filtration, ion exchange and hydroxylapatite chromatography were used for the purification of PAL from lettuce seedlings.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Dissertation

Year

1983

Author dc:creator

• KUDAKASSERIL, GEORGE JOSE

• Biology
• Plant Physiology

Repository record dc:identifier

https://scholars.unh.edu/dissertation/1414

OAI identifier oai:identifier

oai:scholars.unh.edu:dissertation-2413