Microbial L-phenylalanine ammonia-lyase. Purification, subunit  structure and kinetic properties of the enzyme from Rhizoctonia solani.
1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4.  It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.
D000642:Ammonia-Lyases / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D007700:Kinetics; D046911:Macromolecular Substances; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D010650:Phenylalanine Ammonia-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D012232:Rhizoctonia / Q000201:enzymology*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase