The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.

Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.     

Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria

Phenylalanine ammonia-lyase immobilized within semipermeable microcapsules has an assayed enzyme activity which is 20% +/- 4% of the enzyme in free solution. The Km for the immobilized enzyme remained the same as that of the free enzyme. The pH optimum also remained unchanged at pH 8.5 +/- 1.0. At the lower pH range, enzyme activity is higher for the immobilized enzyme. Daily oral administration of microencapsulated phenylalanine ammonia-lyase to phenylketonuric rats decreased the systemic phenylalanine level by 35 +/- 8% in 2 days (P less than 0.05) and by 75 +/- 8% in 7 days (P less than 0.001).     

Phenylalanine and tyrosine ammonia-lyase activity in Sporobolomyces pararoseus

Phenylalanine ammonia-lyase from Sporobolomyces pararoseus was purified more than 450-fold. Polyacrylamide disc gel electrophoresis of this purified enzyme gave a single major protein band. Tyrosine ammonia-lyase activity was monitored during the purification of phenylalanine ammonia-lyase. Deaminating activities for phenylalanine and tyrosine were not separated during the purification process. The existence of one ammonia-lyase with bisubstrate activity is postulated.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.     

Entrapment of phenylalanine ammonia-lyase in silk fibroin for protection from proteolytic attack

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.     

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model

Microencapsulation of the enzyme phenylalanine ammonia-lyase was developed for in vivo depletion of systemic phenylalanine in phenylketonuric rats. Compared to normal rats, systemic phenylalanine blood levels in phenylketonuric rats was increased by 15-20-fold. Daily oral administration of 1 unit of phenylalanine ammonia-lyase-loaded artificial cells to phenylketonuric rats lowered the systemic phenylalanine level to 58% +/- 18% (mean + S.D.) in 7 days (P less than 0.010), while 5 units lowered the systemic phenylalanine level to 25% +/- 8%. 5 units of the immobilized enzyme lowered the systemic phenylalanine level to normal levels within 6 days. Phenylketonuric treated rats showed no signs of abnormal behavior and weight loss compared to phenylketonuric non-treated rats. The immobilized enzyme within artificial cells is therefore protected against low gastrointestinal pH and proteolytic enzymes.     

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.     

Effects of Light and of Fusarium solani on Synthesis and Activity of Phenylalanine Ammonia-Lyase in Peas

Phenylalanine ammonia-lyase was purified from peas, and a specific antiserum against the enzyme was produced in rabbits. The antiserum was used to study the first 8 hours of the phenylalanine ammonia-lyase activity response in two different organs of the pea from different developmental stages and in response to two different stimuli. Etiolated seedlings were pulse-labeled with l-[(35)S]methionine after either no light exposure or after specific periods of irradiation with blue light. Immature pods were pulse labeled with mixed l-[(3)H]amino acids after specific time periods following inoculation of the pod endocarp surfaces with macroconidia of Fusarium solani. Immunoprecipitates isolated from extracts of each group were analyzed with sodium dodecyl sulfate disc gel electrophoresis and were found to contain a radioactive protein with an electrophoretic mobility identical to that of the phenylalanine ammonia-lyase subunit (M(r) 81,000). The radioactivity contained in the subunit band was interpreted as being due to de novo synthesis of the enzyme. The net rate of phenylalanine ammonia-lyase labeling, found to be initially low in both tissue types, rose dramatically, peaking at approximately a six- to ten-fold greater level at 4 hours after the beginning of the stimulus. Thereafter, the rate of labeling declined slowly. Inoculation with F. solani f. sp. pisi, a true pathogen of peas, caused a fifty per cent greater rate of peak labeling than did inoculation with a nonpathogen, F. solani f. sp. phaseoli. The time profile of the changing rate of labeling correlates with the changing activity level of the enzyme which peaks at 12 hours after the onset of the stimulus. The data presented favor a model which explains the changing activity of phenylalanine ammonia-lyase as being due to a changing rate of synthesis or degradation (or both) of the enzyme rather than due to the activation of a preformed zymogen.     

Phenylalanine ammmonia-lyase activity in permeabilised yeast cells (Rhodotorula glutinis)

Summary Whole cells of the yeast Rhodotorula glutinis IFO 0559 had low phenylalanine ammonia-lyase (PAL) activity due to a limited membrane permeability barrier for phenylalanine. Permeabilization with detergents and organic solvents increased cellular PAL activity significantly and of these cetyltrimethylammonium bromide gives the maximum increase (6-fold). PAL activity of such detergent permeabilized cells could be used for the synthesis and degradation of l-phenylalanine.     

Reversible inactivation of phenylalanine hydroxylase by catecholamines in cultured hepatoma cells.

Phenylalanine hydroxylase in Reuber H4 hepatoma cell cultures can be rapidly inactivated by the addition of epinephrine, norepinephrine, dopamine, or 3,4-dihydroxyphenylalanine, in order of decreasing effectiveness, to the culture medium. The enzyme was 50% inactivated in 1 hour by 25 muM (R)-epinephrine or 45 muM (R)-norepinephrine in the medium. High concentrations of epinephrine caused a 70% inactivation in 15 min. Phenylalanine hydroxylase appears to be reversibly inactivated by epinephrine within the cells; since washing the compound off the cell cultures resulted in a rapid restoration of enzyme activity (40% in 1 hour), cycloheximide had little effect on the initial rate of recovery of enzyme activity and the same amount of phenylalanine hydroxylase antigen per cell was isolated from treated and normal cultures. Both (S)- and (R)-epinephrine inactivated the enzyme, and 0.1 mM desmethylimipramine, an inhibitor of amine transport, significantly decreased the effect of epinephrine on the hydroxylase activity. The possibility, suggested by the above results, that epinephrine might be directly inactivating phenylalanine hydroxylase within the cells was supported by the finding that purified rat liver phenylalanine hydroxylase would be 50% inactivated by 1.5 muM epinephrine in 10 min.     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.     

Intracellular Localization of Two Enzymes Involved in Coumarin Biosynthesis in Melilotus alba

The localization of phenylalanine ammonia-lyase [EC 4.3.1.5] within sweet clover (Melilotus alba) leaves was investigated. Apical buds and axillary leaves contained 15 to 30 times more enzyme activity than did mature leaves. Mesophyll protoplasts were prepared by digesting young leaves with Cellulysin and Macerase and were gently ruptured yielding intact chloroplasts. These chloroplast preparations exhibited neither phenylalanine ammonia-lyase nor o-coumaric acid O-glucosyltransferase activities. The general enzymic properties of sweet clover leaf phenylalanine ammonia-lyase were similar to those described for this enzyme isolated from other plant species. The conversion of l-phenylalanine to trans-cinnamic acid, which occurred at an optimum pH of about 8.7, was strongly inhibited by the metabolites trans-cinnamic and o-coumaric acids. In contrast, o-coumaric acid glucoside, coumarin, p-coumaric acid, and melilotic acid had no significant effect on the reaction rate.     

Phenylalanine ammonia-lyase and chalcone synthase in glands ofPrimula kewensis (W. Wats): immunofluorescence and immunogold localization

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were localized by indirect immunofluorescence and immunogold labeling in glands ofPrimula kewensis. Both enzymes were exclusively present in the head cells of the glands. Phenylalanine ammonialyase was located in the regions of the dense tubular endoplasmic reticulum and occasionally found in more or less spherical organelles that have not yet been identified. Furthermore, an appreciable proportion of the enzyme protein was associated with the plasmalemma and the cell wall of the head cell. In contrast, the occurrence of CHS was restricted to the spherical, unidentified cell compartments. Our findings indicate that the gland cells have the potential for flavonoid biosynthesis. When a mutant ofP. kewensis forming structurally intact glands but incapable of farina excretion was studied, neither PAL nor CHS were found in the head cells.Abbreviations CHS chalcone synthase - IgG immunoglobulin G - PAL phenylalanine ammonia-lyase Financial support from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged. We are grateful to Mrs. Karin Schlattmann and Mrs. Susanne Otter for preparing the ultrathin sections and to Mrs. Marianne Opalka for taking the photographs.     

Relationship of Phenylalanine Ammonia-lyase Activity to o-Hydroxycinnamic Acid Content in Melilotus alba

Phenylalanine ammonia-lyase activity was investigated in preparations representing various parts of sweetclover (Melilotus alba Desr.) plants of CuCu and cucu genotypes. In contrast to other plant parts, very young leaves and stems of CuCu plants displayed high phenylalanine ammonia-lyase activity. Initial leaf samples from CuCu plants were approximately 3 times as high in enzyme activity as leaves from cucu plants, but stems were only slightly higher in activity. Defoliation of the plants resulted in decreased enzyme activity, increased o-hydroxycinnamic acid content, and essentially no difference in enzyme activity between the genotypes. It appears that phenylalanine ammonia-lyase activity in leaves is not primarily controlled by the Cu/cu alleles and that the reaction catalyzed by this enzyme is not the limiting step in o-hydroxycinnamic acid synthesis.     

The inactivation of phenylalanine hydroxylase by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and the aerobic oxidation of the latter. The effects of catalase, dithiothreitol and reduced nicotinamide–adenine dinucleotide

1. Phenylalanine hydroxylase is inhibited by its cofactor, 6,7-dimethyltetrahydropterin. The rate of inactivation, which is irreversible, increases with the concentration of cofactor. 2. Catalase, in sufficient amount relative to cofactor, prevents this inactivation. More tyrosine is formed in the presence of added catalase. 3. Dithiothreitol in the presence of liver extract also prevents inactivation of the enzyme by the cofactor and stimulates hydroxylation of phenylalanine, probably by protecting the cofactor from oxidation and regenerating it from a dihydropterin reaction product. Dithiothreitol restores linearity of rate at very low enzyme concentrations. 4. Dimethyltetrahydropterin is unstable when the solution is exposed to air but is stabilized by dithiothreitol the aerobic oxidation of which is greatly accelerated by dimethyltetrahydropterin. 5. NADH together with liver extract stabilizes the cofactor but not phenylalanine hydroxylase. 6. It is suggested that either hydrogen peroxide or an organic peroxide formed by oxidation in air of the cofactor is the substance attacking phenylalanine hydroxylase, dithiothreitol and cofactor.     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Some properties of polyethylene glycol:phenylalanine ammonia-lyase adducts.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to phenylalanine ammonia-lyase from Rhodotorula glutinis. Attachment of sufficient quantities of PEG to phenylalanine ammonia-lyase substantially reduces immunological recognition and clearance of the conjugated enzyme in mice. The modified enzyme demonstrates altered catalytic properties such as shifts in the pH and temperature optima, an increase in the Michaelis-Menten constant, and a lowered Vmax in comparison with the native enzyme. PEG-phenylalanine ammonia-lyase has increased resistance to proteolytic digestion, particularly when in the presence of cinnamate, a competitive inhibitor, while the native enzyme is rapidly inactivated. In the ultracentrifuge PEG-phenylalanine ammonia-lyase exhibits a lower sedimentation rate than the unmodified enzyme, despite the fact that it is much larger. The electrophoretic mobility of PEG-phenylalanine ammonia-lyase is greatly decreased in comparison to the unmodified enzyme. PEG-phenylalanine ammonia-lyase had a much longer blood-circulating life in mice, both initially and after a number of injections, than did the native enzyme. PEG-phenylalanine ammonia-lyase was a good immunogen but a poor antigen in mice and rabbits, that is, it readily induced antibody formation, but reacted poorly in vitro with the antibodies that were formed against it.     

Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase from Rhodosporidium toruloides

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.     

The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.