| dc.description.abstract | Rhizoctonia solani employs two pathways, viz., phenylpyruvate pathway and cinnamate pathway, to degrade L-phenylalanine. The intermediates in the phenylpyruvate pathway are phenylacetate, o-hydroxyphenylacetate, and m-hydroxyphenylacetate. m-Hydroxyphenylacetate, which is not further metabolized by this organism, is excreted into the medium. In the cinnamate route, L-phenylalanine is converted into cinnamate, o-coumarate, p-coumarate, p-hydroxybenzoate, and protocatechuate. Optimal levels of PAL were obtained when R. solani was grown on a synthetic medium containing O_2 (0.5%), glucose (5.5%), and L-phenylalanine (0.3%) for 4.5 days.
PAL was purified from the acetone-dried powders of the mycelial felts of R. solani. A useful modification in protamine sulfate treatment, to get substantial purification of the enzyme in a single step, is described. The enzyme is homogeneous on polyacrylamide gel electrophoresis, analytical ultracentrifugation, and by Ouchterlony double-diffusion technique. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. It is sensitive to carbonyl reagents, and the inhibition is not reversed by gel filtration.
It is stable to heating at 60°C for 1 hr. The molecular weight of the enzyme, as determined by Sephadex G-200 chromatography, thin-layer gel filtration, and sucrose density gradient centrifugation, is around 350,000. The sedimentation constant and the Stokes radius for this enzyme are estimated as 13.5 S and 62 Å by analytical ultracentrifugation and molecular sieve chromatography, respectively. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70,000 ( ) and 90,000 ( ), respectively. Amino acid analysis has shown that the enzyme is rich in aspartic and glutamic acids.
Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. The double reciprocal plots are biphasic (concave downwards), and Hofstee plots show a curvilinear pattern. The apparent K_m 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, V_max increases by 2- to 2.5-fold. The value of the Hill coefficient is 0.5.
Steady-state rates of PAL reaction in the presence of inhibitors like L-phenylalanine, cinnamic acid, p-coumaric acid, caffeic acid, dihydrocaffeic acid, 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 Rhizoctonia PAL. The enzyme could not be desensitized by treatment with HgCl_2, 2-chloromercuribenzoate, or by repeated freezing and thawing.
Detailed studies were carried out on the regulation of PAL from R. solani, and the properties were compared with the enzyme from higher plants. PAL activity is observed only when phenylalanine is present in the medium. Unlike the enzyme from higher plants, it is not affected by light, auxins, or temperature. Among the various amino acids and structural analogs tested, only the aromatic amino acids are effective in inducing the enzyme.
PAL is known to exist as an inactive pool in some cases in higher plants, and in the present case, the appearance of enzyme activity is inhibited both by actinomycin D and cycloheximide. Conclusive evidence for de novo synthesis of PAL in R. solani is obtained by incorporating ^14C-labeled amino acids into the enzyme during induction. Electrophoretic analysis of the purified enzyme shows a single protein band which coincides with the radioactive peak and enzyme activity. There is no indication for the presence of inactive PAL in R. solani during uninduced conditions, as tested by Ouchterlony double-diffusion technique using the antiserum raised against purified PAL.
Glucose, intermediates of the TCA cycle like citric acid, -ketoglutaric acid, succinic acid, and end products of phenylalanine degradation like p-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid repress the synthesis of PAL. The induction of PAL depends on the concentration of the inducer. Phenylalanine is required for both synthesis and maintenance of the enzyme levels. Inactivation of the enzyme is hastened in the absence of the inducer. The half-life of PAL is around 2 hr. The process of inactivation seems to involve proteolytic degradation of the enzyme rather than mere loss of catalytic power. | |
