Studies on the phenylalanine hydroxylase system in liver slices.

A method was developed to study the unsupplemented phenylalanine hydroxylase system in rat liver slices. All of the components of the system--tetrahydrobiopterin, dihydropteridine reductase, and the hydroxylase itself--are present under conditions which should be representative of the actual physiological state of the animal. The properties of the system in liver slices have been compared to those of the purified enzyme in vitro. The three pterins, tetrahydrobiopterin, 6,7-dimethyltetrahydropterin, and 6-methyltetrahydropterin, all stimulate the hydroxylation of phenylalanine when added to the liver slice medium in the presence of a chemical reducing agent. The relative velocities found at 1 mM phenylalanine and saturating pterin concentrations are: tetrahydrobiopterin, 1; 6,7-dimethyltetrahydropterin, 2.5; 6-methyltetrahydropterin, 13. This ratio of activities is similar to that found for the purified, native phenylalanine hydroxylase and indicates that the enzyme in vivo is predominantly in the native form. Rats pretreated with 6-methyltetrahydropterin showed enhanced phenylalanine hydroxylase activity in liver slices demonstrating for the first time that an exogenous tetrahydropterin can interact with the phenylalanine hydroxylase system in vivo. This finding opens up the possibility of treating phenylketonurics who still possess some residual phenylalanine hydroxylase activity with a tetrahydropterin like 6-methyltetrahydropterin which can give a large increase in rate over that seen with the natural cofactor, tetrahydrobiopterin.     

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

Effect of glucagon on hepatic phenylalanine hydroxylase in vivo

Moderate doses of glucagon (20 g/kg I.V.) are sufficient to stimulate rat hepatic phenylalanine hydroxylase in vivo. In addition, the stimulation of the tetrahydrobiopterin-dependent phenylalanine hydroxylase activity in livers of animals fed on a high-protein diet has been correlated with an elevated phosphate content. The tetrahydrobiopterin-dependent hydroxylase activity in these animals can be further elevated by glucagon-stimulated phosphorylation. These results indicate that physiological changes in glucagon concentration modulate rat liver phenylalanine hydroxylase activity in vivo. The current understanding of the role of phosphorylation in regulating human phenylalanine hydroxylase is also considered.     

Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

Phenylalanine-induced phosphorylation and activation of rat hepatic phenylalanine hydroxylase in vivo.

Rats were given intraperitoneal injections of 2 mCi of carrier-free 32Pi and substances known to activate liver phenylalanine hydroxylase. After 30 min, these animals were anesthetized and their livers removed for analysis of enzyme activity, 32Pi incorporation into immunoprecipitated phenylalanine hydroxylase and [gamma-32P]ATP specific activity. Following glucagon treatment, rat liver phenylalanine hydroxylase activity was stimulated more than 6-fold when assayed in the presence of the natural cofactor, tetrahydrobiopterin (BH4). Glucagon injection also resulted in an incorporation of 0.41 mol of 32Pi/mol of hydroxylase subunit (approximately 50,000 Da). In vivo stimulation of phenylalanine hydroxylase activity and 32Pi incorporation by glucagon had been previously observed in this laboratory (Donlon, J., and Kaufman, S. (1978) J. Biol. Chem. 253, 6657-6659). However, we show for the first time in the present study that in vivo treatment with phenylalanine alone results in a 4-fold increase in the BH4-dependent activity of phenylalanine hydroxylase concomitant with a significant incorporation of phosphate into phenylalanine hydroxylase (0.51 mol of 32Pi/mol of hydroxylase subunit). It is further demonstrated in vivo that the combined treatment with phenylalanine and glucagon results in a greater than 10-fold stimulation of BH4-dependent activity and the greatest level of 32Pi incorporation (0.75 mol of 32Pi/mol of hydroxylase subunit). Phenylalanine did not produce an elevation in plasma glucagon in these animals. A model is, thereby, proposed with respect to the ligand binding effects of phenylalanine on the state of phosphorylation and activation of phenylalanine hydroxylase. The significance of these regulatory roles are considered in light of the probable physiological environment of the enzyme.     

Effects of experimental diabetes on rat hepatic phenylalanine hydroxylase in vivo

1. The stimulated levels of phenylanine hydroxylase activity in liver extracts from streptozotocin-induced diabetic rats (Donlon and Beirne, 1982) have been correlated with an increased rate of phenylalanine catabolism in vivo. 2. The levels of hepatic phenylalanine hydroxylase protein in diabetic rats become elevated. This effect is not seen in diabetic animals concurrently treated with insulin. 3. The rate of synthesis of liver phenylalanine hydroxylase in 5-day diabetics is 260% that of control animals. 4. These observations are discussed with reference to the regulation of hepatic phenylalanine hydroxylase and phenylanine metabolism in rats.     

Studies on the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase

The uncoupled portion of the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase can be described by the same model as we have recently derived for the fully uncoupled reaction (Davis, M.D. and Kaufman, S. (1989) J. Biol. Chem.264, 8585–8596). Although essentially no hydrogen peroxide is formed during the fully coupled oxidation of tetrahydrobiopterin or 6-methyltetrahydropterin by phenylalanine hydroxylase when phenylalanine is the amino acid substrate, significant amounts of hydrogen peroxide are formed during the partially uncoupled oxidation of 6-methyltetrahydropterin whenpara-fluorophenylalanine orpara-chlorophenylalanine are used in place of phenylalanine. Similarly, during the partially uncoupled oxidation of the unsubstituted pterin, tetrahydropterin, even in the presence of phenylalanine, hydrogen peroxide formation is detected. The 4a-carbinolamine tetrahydropterin intermediate has been observed during the fully uncoupled tyrosine-dependent oxidations of tetrahydropterin and 6-methyltetrahydropterin by lysolecithin-activated phenylalanine hydroxylase, suggesting that this species is also a common intermediate for uncoupled oxidations by this enzyme.Abbreviations BH₄ 6-[dihydroxypropyl-(L-erythro)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) - 6MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PH₄ 5,6,7,8-tetrahydropterin - BH₃OH 4a-hydroxytetrahydropterin (4a-carbinolamine) - qBH₂ quinonoid dihydrobiopterin - q6MPH₂ quinonoid dihydro-6-methylpterin - qPH₂ quinoid dihydropterin - PAH phenylalanine hydroxylase - DHPR dihydropteridine reductase - PHS phenylalanine hydroxylase stimulating enzyme which is 4a-carbinolamine dehydratase - SOD superoxide dismutase - HPLC high performance liquid chromatography - R.T. retention time Special issue dedicated to Dr. Santiago Grisolia.     

New assay for pregnenolone and progesterone 17alpha- hydroxylase based on the specific substitution of a tritium situated on carbon 17.

A new assay for the measurement of steroid 17alpha-hydroxylase activity in beef adrenals is described. This method is based on the biochemical mechanism of the enzymic reaction, i.e. the direct and stereospecific substitution of the proton located on the hydroxylated position. Progesterone or pregnenolone specifically labelled on the 17 position are solubilized in the incubation mixture with the help of Tween 80 and incubated under optimal conditions. The tritium enzymically released from the substrate is found in the medium as a molecule of water which is then distilled under reduced pressure and counted by liquid scintillation. The results obtained with this new method are comparable with those obtained with a conventional method using a 14C-labelled substrate.     

The activity of 2,4,5-triamino-6-hydroxypyrimidine in the phenylalanine hydroxylase system.

The pyrimidine moiety of a pterin, 2,4,5-triamino-6-hydroxypyrimidine, has been found to be active in the phenylalanine-hydroxylating system. The phenylalanine-dependent, phenylalanine hydroxylase-catalyzed reaction in the presence of the pyrimidine is largely, but not completely, uncoupled; the ratio of DPNH oxidized to tyrosine formed is about 20 to 1. In addition to the pyrimidine having activity with phenylalanine hydroxylase, a product of the pyrimidine is also a substrate for dihydropteridine reductase. The activity of the pyrimidine with the hydroxylase indicates that neither carbon atoms 6 or 7 of the pterin ring is involved in activation of oxygen during the hydroxylase-catalyzed reaction.     

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.     

Theoretical foundation and experimental proof of the accumulating transfer of tritium from water into DNA and other biomolecules in vitro and in vivo

Heisenberg's uncertainty principle, which describes a fundamental property of matter, predicts the light hydrogen isotope to prefer the strong hydrogen bridge positions (with large local uncertainty). In contrast, the heavy hydrogen isotope tritium originally localized in water, should finally be found in the exchangeable hydrogen bridge positions of proteins, carbohydrates and nucleotides which are definitely less strong than those of water. The fractionation factor of tritium and protium measured between water and DNA as well as the mixture of biomolecules of liver confirm this conclusion and show in addition, a tritum accumulation also in the hydration sheets of the biomolecules. Furthermore, the larger intrinsic rate of natural increase of tritium than of hydrogen in tissue solids of growing maize and barley confirm the accumulating tritium transfer from water to biomolecules taking place in vivo.     

Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase.

NMR spectroscopy and X-ray crystallography have provided important insight into structural features of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). Nevertheless, significant problems such as the substrate specificity of PAH and the different susceptibility of TH to feedback inhibition by l-3,4-dihydroxyphenylalanine (l-DOPA) compared with dopamine (DA) remain unresolved. Based on the crystal structures 5pah for PAH and 2toh for TH (Protein Data Bank), we have used molecular docking to model the binding of 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the substrates phenylalanine and tyrosine to the catalytic domains of PAH and TH. The amino acid substrates were placed in positions common to both enzymes. The productive position of tyrosine in TH.BH4 was stabilized by a hydrogen bond with BH4. Despite favorable energy scores, tyrosine in a position trans to PAH residue His290 or TH residue His336 interferes with the access of the essential cofactor dioxygen to the catalytic center, thereby blocking the enzymatic reaction. DA and l-DOPA were directly coordinated to the active site iron via the hydroxyl residues of their catechol groups. Two alternative conformations, rotated 180 degrees around an imaginary iron-catecholamine axis, were found for DA and l-DOPA in PAH and for DA in TH. Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction product l-DOPA to TH, thereby saving the enzyme from direct feedback inhibition.     

Effect of serum on phenylalanine hydroxylase levels in cultured hepatoma cells.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.     

Incorporation of tritium into cell materials of Rhodopseudomonas spheroides from tritiated water in the medium under aerobic conditions.

When Rhodopseudomonas spheroides cells grown aerobically in the dark were incubated in medium containing tritiated water (THO), incorporation of T into the bacterial cell materials occurred under growth and no-growth conditions. The overall T incorporation under no-growth conditions was stimulated by vigorous aeration and was suppressed strongly in the presence of either 10(-3) M KCN or 0.3% HgCl2, indicating that the bulk of the incorporation might depend upon bacterial cell metabolism or respiration. 10 mug/ml chloramphenicol and 20 mug/ml rifamipicin slightly suppressed the T incorporation. The extent of T incorporation was proportional to the concentration of T in the medium. Accordingly, regardless of differences in the concentration of T in the medium, the maximum ratio of T content per hydrogen atom in the cell materials to that of THO in the medium was approximately 0.2 in non-growing cells and 0.5 in growing cells, whereas the value was 0.02-0.03 in cells incubated in medium containing KCN or HgCl2. The non-growing cells aerated in THO medium were lyophilized and fractionated by the modified method of Schneider. More than 40% of the total T incorporated into the cell materials was recovered in the cold PCA-soluble fraction, whereas the distribution of T into fractions solbule in ether-ethanol, hot PCA and alkali was 10 to 20% each. More than 75% of the T extracted in the cold PCA-soluble fraction was volatile. While the amounts of RNA and protein in the non-growing cells decreased on adding chloramphenicol or rifampicin, the distribution of T in these fractions did not change much. Our results on T incorporation into non-growing cells indicate that the major T incorporation into bacterial cell materials is independent of biosynthetic reactions using labeled precursors produced by the assimilation of T into metabolites, but presumably depends on energy-linked conformational changes of macromolecules.     

The regulation of phenylalanine hydroxylase in rat tissues in vivo. The maintenance of high plasma phenylalanine concentrations in suckling rats: a model for phenylketonuria.

Maximum inhibition of phenylalanine hydroxylase activity in the liver (85%) and in the kidney (50%) of suckling rats required the administration of over 9 mumol of p-chlorophenylalanine/10g body weight. Despite the decrease in the total activity from 184 to 34 units per 10g body weight, the injection of as much as 26 mumol of phenylalanine was required for its concentration in plasma to be still considerably elevated 12h later. In rats injected with p-chlorophenylalanine every 48h and with phenylalanine every 24h from 3 to 18 days of age, the hepatic and renal phenylalanine hydroxylase remained inhibited, whereas the activities of three other hepatic enzymes were unchanged. There was about 20% inhibition of brain and body growth, but no interference with the developmental formation of several cerebral enzymes (four dehydrogenases, hexokinase and glutaminase) was detected. In the course of this prolonged treatment, the phenylalanine concentrations in plasma increased gradually; on day 2 and day 8 (measured 12h after the last injection) they were 800 and 1395 nmol/ml respectively; on day 15, 12 and 18h after the usual injection, the values were 2030 and 1030 respectively as opposed to the 96 nmol in untreated rats. This degree of hyperphenylalaninaemia, persisting for 18h per day throughout a critical period of development, fulfils the primary criterion of a suitable animal model for phenylketonuria.     

Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase.

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.