Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli

A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.     

Effect of alkaline pH on the activity of rat liver phenylalanine hydroxylase

The pH optimum of rat liver phenylalanine hydroxylase is dependent on the structure of the cofactor employed and on the state of activation of the enzyme. The tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase has a pH optimum of about 8.5. In contrast, the 6,7-dimethyltetrahydropterin-dependent activity is highest at pH 7.0. Activation of phenylalanine hydroxylase either by preincubation with phenylalanine or by limited proteolysis results in a shift of the pH optimum of the tetrahydrobiopterin-dependent activity to pH 7.0. Activation of the enzyme has no effect on the optimal pH of the 6,7-dimethyltetrahydropterin-dependent activity. The different pH optimum of the tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase is due to a change in the properties of the enzyme when the pH is increased from pH 7 to 9.5. Phenylalanine hydroxylase at alkaline pH appears to be in an altered conformation that is very similar to that of the enzyme which has been activated by preincubation with phenylalanine as determined by changes in the intrinsic protein fluorescence spectrum of the enzyme. Furthermore, phenylalanine hydroxylase which has been preincubated at an alkaline pH in the absence of phenylalanine and subsequently assayed at pH 7.0 in the presence of phenylalanine shows an increase in tetrahydrobiopterin-dependent activity similar to that exhibited by the enzyme which has been activated by preincubation with phenylalanine at neutral pH. Activation of the enzyme also occurs when m-tyrosine or tryptophan replace phenylalanine in the assay mixture. The predominant cause of the increase in activity of the enzyme immediately following preincubation at alkaline pH appears to be the increase in the rate of activation by the amino acid substrate. However, in the absence of substrate activation, phenylalanine hydroxylase preincubated at alkaline pH displays an approximately 2-fold greater intrinsic activity than the native enzyme.     

DL-苯丙氨酸酶法拆分

ＤＬ－苯丙氨酸的拆分是以Ｎ－乙酰－ＤＬ－苯丙氨酸铵为起始原料,经猪肾酰化酶不对称水解作用,再经离子交换色谱分离,减压浓缩得到Ｌ－苯丙氨酸和Ｎ－乙酰－Ｄ－苯丙氨酸结晶。     

The PKU locus in man is on chromosome 12

Classical phenylketonuria (PKU) is a typical example of inborn errors in metabolism and is characterized by a complete lack of the hepatic enzyme phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine. The genetic disorder causes impairment of postnatal brain development, resulting in severe mental retardation in untreated children. The disease is transmitted as an autosomal recessive trait and has a collective prevalence of about one in 10,000 among Caucasians, so that 2% of the population are carriers of the PKU trait. We have recently reported the cloning of human phenylalanine hydroxylase cDNA and that the human chromosomal phenylalanine hydroxylase gene is encoded by a unique DNA sequence. Using the human phenylalanine hydroxylase cDNA clone to analyze a clonal human/mouse hybrid cell panel by Southern hybridization, the phenylalanine hydroxylase gene has been assigned to human chromosome 12. Since the hypothesis that classical PKU is caused by structural mutations in the phenylalanine hydroxylase gene itself rather than through some transregulatory mechanisms has recently been confirmed by gene mapping, the PKU locus in man is determined to be on chromosome 12.     

The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.     

Misreading of the argI message in Escherichia coli

It has previously been shown that either phenylalanine codon, UUU or UUC, could be misread as leucine during phenylalanine starvation, if the codons encoded residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase (OTC). However, no leucine misincorporation was detected when either of these same codons encoded residue 3. Here we report that leucine misincorporation can be directed by a UUU codon for residue 3 of OTC during phenylalanine starvation, if the argI gene has been mutated so that the codon preceding the UUU has been changed from the rarely used glycine codon GGG to the more commonly used GGC.     

Photoluminescence band and anti-Stokes emission of native collagen in the visible range of the spectrum]

Photoluminescence (PL) of collagen, phenylalanine and tyrosine has been experimentally studied in the range of 400-700 nm. Anti-Stokes emission was observed when PL was excited by visible light. Possible participation of an excimer in generation of PL was shown. It has been pointed out that phenylalanine chromophore plays a dominant role in fluorescence generation of collagen in the spectrum visible range.     

Studies on the phenylalanine hydroxylase system in vivo. An in vivo assay based on the liberation of deuterium or tritium into the body water from ring-labeled L-phenylalanine.

The rate of release of deuterons into the body water from 2,3,4,5,6-pentadeutero-L-phenylalanine has been shown to be a valid measure of the activity of the phenylalanine hydroxylase system in vivo. At a dose of 0.5 g/kg, the rate of release of deuterons is linear for 60 to 90 min. Male rats, which had previously been shown to have 22 to 25% more phenylalanine hydroxylase activity in liver extracts than female rats, produced deuterons from deuterated phenylalanine at a rate 20 to 30% greater than female rats. p-Chlorophenylalanine, which irreversibly inhibits phenylalanine hydroxylase in vivo, caused a similar degree of inhibition of the rate of deuteron formation as was found when phenylalanine hydroxylase was measured in extracts from the same group of animals. Methotrexate, which inhibits the phenylalanine hydroxylase system by preventing regeneration of the tetrahydropteridine cofactor, caused parallel inhibition of the in vivo assay as well as when the conversion of phenylalanine to tyrosine was measured in liver slices. Randomly ring-tritiated phenylalanine can be used interchangeably with ring-deuterated phenylalanine if greater sensitivity is needed in the in vivo assay for phenylalanine hydroxylase. However, a dose of 20 to 30 muCi/kg is required. The in vivo deuterium release assay described in this paper should be useful in studying the physiological control of the phenylalanine hydroxylating system. It also may be of value in differentiating between individuals who are heterozygotes for phenylketonuria and those who are homozygotes for hyperphenylalaninemia.     

Evaluation of the phenylalanine tolerance test in detection of heterozygote carriers of phenylketonuria gene]

The study aimed at identifying heterozygotic phenylketonuria gene carriers with phenylalanine tolerance test performed in Lublin region. Serum phenylalanine concentration has been assayed during fasting and 1 and 2 hours following oral phenylalanine load in the dose of 0.1 g per 1 kg body weight. The study involved 203 individuals of the general population and 29 heterozygotes with phenylketonuria gene. Blood serum phenylalanine was assayed with Guthrie' technique. Statistical analysis has shown that hyperphenylalaninemia is relatively frequent in fasting individuals of the general population (59.1%). The same was demonstrated in 5 heterozygotes. Phenylalanine tolerance test did not allow to identify heterozygotic carries of phenylketonuria gene in the general population though fasting and after phenylalanine load increased blood serum levels of this amino acid are a criterium of hyperphenylalaninemia in the group of tested individuals (29%).     

Interaction with a monoclonal antibody alters the expression of co-operativity by phenylalanine hydroxylase from rat liver.

Phenylalanine hydroxylase purified from rat liver shows positive co-operativity in response to variations in phenylalanine concentration when assayed with the naturally occurring cofactor tetrahydrobiopterin. In addition, preincubation of phenylalanine hydroxylase with phenylalanine results in a substantial activation of the tetrahydrobiopterin-dependent activity of the enzyme. The monoclonal antibody PH-1 binds to phenylalanine hydroxylase only after the enzyme has been preincubated with phenylalanine and is therefore assumed to recognize a conformational epitope associated with substrate-level activation of the hydroxylase. Under these conditions, PH-1 inhibits the activity of phenylalanine hydroxylase; however, at maximal binding of PH-1 the enzyme is still 2-3 fold activated relative to the native enzyme. The inhibition by PH-1 is non-competitive with respect to tetrahydropterin cofactor. This suggests that PH-1 does not bind to an epitope at the active site of the hydroxylase. Upon maximal binding of PH-1, the positive co-operativity normally expressed by phenylalanine hydroxylase with respect to variations in phenylalanine concentration is abolished. The monoclonal antibody may therefore interact with phenylalanine hydroxylase at or near the regulatory or activator-binding site for phenylalanine on the enzyme molecule.     

A monoclonal antibody to aromatic amino acid hydroxylases. Identification of the epitope.

PH8 monoclonal antibody has previously been shown to react with all three aromatic amino acid hydroxylases, being particularly useful for immunohistochemical staining of brain tissue [Haan, Jennings, Cuello, Nakata, Chow, Kushinsky, Brittingham & Cotton (1987) Brain Res. 426, 19-27]. Western-blot analysis of liver extracts showed that PH8 reacted with phenylalanine hydroxylase from a wide range of vertebrate species. The epitope for antibody PH8 has been localized to the human phenylalanine hydroxylase sequence between amino acid residues 139 and 155. This highly conserved region of the aromatic amino acid hydroxylases has 11 out of 17 amino acids identical in phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase.     

Binding site specificity of gamma-radiation-induced crosslinking between phenylalanine and a phenylalanine-containing tetrapeptide

The binding site specificity of crosslinking mediated by the hydroxyl radical has been investigated in a simple model system: a tetrapeptide, Gly-Gly-Phe-Leu, and 14C-labeled phenylalanine. Crosslinking leads to the tetrapeptide-phenylalanine adduct which has been isolated by gel filtration. The amino acid analysis of these adducts compared with those of gamma-radiation-induced dimers of the tetrapeptide and of the dipeptide, Gly-Phe, shows that only the phenylalanine residue is affected and that the same new peaks appear in each case. Spectrophotometric measurement indicates that the extinction coefficient at 260 nm of dimeric tetrapeptide is four times higher than that of monomeric, as is dimeric phenylalanine compared to monomeric. These observations suggest a common crosslinking mechanism in all three cases that involves the aromatic ring of phenylalanine. The appearance of several radioactive peaks in the gel filtration separation of the acid hydrolysate of the adduct suggests that the crosslinking involves more than one possible modification of the phenylalanine. Three distinct tetrapeptide-Phe species, corresponding to molecular weights of 555, 573, and 591, were observed by fast atom bombardment mass spectrometry. The partial release of radioactive phenylalanine from the tetrapeptide-phenylalanine adducts by acid hydrolysis indicates the liability of some phenylalanine-phenylalanine bonds.     

A possible role of divalent manganese ions in the photoinduction of phenylalanine ammonia-lyase

Divalent Mn ions cause an increase in the level of phenylalanine ammonia-lyase in gherkin hypocotyls. With the exception of Mg ions, which had a small effect, no other metal ion has so far been found which could replace the Mn ion in this respect. Invertase and peroxidase were not significantly affected by the Mn treatment. The increase in phenylalanine ammonialyase activity is explained by the removal, under the influence of Mn ions, of hydroxycinnamic acids, which cause repression of phenylalanine ammonia-lyase synthesis and/or inactivation of phenylalanine ammonia-lyase. Arguments are advanced for the hypothesis that photochemical transformations of Mn complexes are involved in the photoinduction of phenylalanine ammonia-lyase in dark-grown gherkin seedlings.     

On the phosphate content of rat liver phenylalanine hydroxylase purified by hydrophobic chromatography

Rat liver phenylalanine hydroxylase purified by hydrophobic chromatography has been found to contain significantly less protein-bound phosphate than enzyme purified by more conventional procedures. Studies with purified hydroxylase of defined phosphate content suggest that phosphorylated species of phenylalanine hydroxylase possess a higher affinity for the hydrophobic matrix than does the non-phosphorylated form. This selectivity may account for the lower phosphate content in phenylalanine hydroxylase purified by hydrophobic chromatography.     

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Binding studies with the allosteric effector phenylalanine.

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.     

Enzymatic Determination of l-Phenylalanine and l-Tyrosine

An enzymatic method for the determination of phenylalanine and tyrosine has been described. This method is based on the formation of cinnamic acid from phenylalanine or the formation of p-coumaric acid from tyrosine by phenylalanine ammonia-lyase of Rhodotorula. Cinnamic acid and p-coumaric acid, which are formed in stoichiometric amounts, are determined spectrophotometrically. Other amino acids and d-isomers of phenylalanine and tyrosine have no effect on this determination.     

Localization of mouse phenylalanine hydroxylase locus on chromosome 10

Mouse phenylalanine hydroxylase has been localized on chromosome 10C2----D1 by in situ hybridization using a mouse phenylalanine hydroxylase cDNA clone. This locus is distinct from the hyperphenylalaninemia locus on chromosome 14 and the locus for tyrosine hydroxylase on chromosome 7.     

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.     

The importance of transamination and decarboxylation in phenylalanine metabolism in vivo in the rat

The extent of hydroxylation, transamination, and decarboxylation in the metabolism of excess phenylalanine in vivo has been examined by measuring the amount of radioactivity from [¹⁴C]phenylalanine that is converted to ¹⁴CO₂ and urinary metabolites. Transamination and direct decarboxylation represent only 6% of total phenylalanine metabolism. The major aromatic metabolites in the urine after phenylalanine loading are phenylacetylglycine, phenylacetic acid, phenylpyruvate, and phenylalanine. A small but significant portion (1.5%) of phenylalanine is degraded to nonaromatic compounds. The maximum phenylalanine oxidation in vivo is approximately $\text{75\%}\text{24}$ h at saturating concentrations of phenylalanine; thus, the major route of degradation of phenylalanine in the rat, even when intake is high, is via formation of acetoacetic acid and fumaric acid.     

The need for high levels of phenylalanine in the diet of Schistocerca gregaria nymphs

It has been demonstrated that an apparently good, nutrient-rich plant can be relatively short of phenylalanine for fifth-instar nymphs of Schistocerca gregaria. Supplementary phenylalanine invariably increased the efficiency of conversion of ingested food to body substance, showing that this was the limiting nutrient. Relatively large quantities of phenylalanine are required for cuticle protein over a few days in the first half of the instar, which is when this amino acid is at a premium.