Mechanism of phenylalanine regulation of phenylalanine hydroxylase

The mechanism of phenylalanine regulation of rat liver phenylalanine hydroxylase was studied. We show that phenylalanine "activates" phenylalanine hydroxylase, converting it from an inactive to active form, by binding at a true allosteric regulatory site. One phenylalanine molecule binds per enzyme subunit; it remains at this site during catalytic turnover and, while there, cannot be hydroxylated. Loss of phenylalanine from the site causes a loss of enzymatic activity. The rate of loss of activation is dramatically slowed by phenylalanine, which kinetically "traps" activated enzyme during relaxation from the activated to unactivated state. An empirical equation is presented which allows calculation of relaxation rates over a wide range of temperatures and phenylalanine concentrations. Kinetic trapping by phenylalanine is a novel effect. It was analyzed in detail, and its magnitude implied that phenylalanine activation involves cooperativity among all four subunits of the enzyme tetramer. A regulatory model is presented, accounting for the properties of the phenylalanine activation reaction in the forward and reverse directions and at equilibrium. Fluorescence quenching studies confirmed that activation increases the solvent accessibility of the enzyme's tryptophan residues. Physical and kinetic properties of purified phenylalanine hydroxylase from rat, rabbit, baboon, and goose liver were compared. All enzymes were remarkably alike in catalytic and regulatory properties, suggesting that control of this enzyme is similar in mammals and birds.     

Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.

The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.     

Effects of phenylalanine and analogues of methionine and phenylalanine on the composition of wool and mouse hair

Administration of the methionine analogue methoxinine (O-methyl-DL-homoserine) to sheep substantially changed the composition of wool; in addition wool fibres were weakened and the staple crimp frequency was reduced for a prolonged period. The proportions of high-tyrosine proteins were reduced by 40-45% whereas the high-sulfur proteins were usually slightly increased. The content of high-tyrosine proteins in wool was still depressed in most sheep 70 days after dosing with methoxinine. These experiments supported a previous finding that the cystine content of wool and its crimp frequency are not causally related. Ethionine, another methionine analogue, did not consistently change the composition of wool. In some sheep there was no change in the proportions of high-tyrosine proteins following administration of ethionine, even though weak wool was produced. This result, together with the lack of association between the content of high-tyrosine proteins and the strength of wool fibres in a sheep given methoxinine plus methionine, indicates that a reduction of the high-tyrosine proteins is not a prerequisite for the production of weak wool. Neither a threefold increase in the phenylalanine intake by mice nor the administration of three analogues of phenylalanine (4-fluoro-DL-phenylalanine, 4-chloro-DL-phenylalanine and beta-(2-thienyl)-DL-alanine) to sheep altered the composition of hair or wool. Fluorophenylalanine was incorporated into all the constituent proteins of wool to the extent of c. 2% of phenylalanine residues. The other analogues studied could not be detected in wool.     

A solvent effect on the side-chain conformation of phenylalanine derivatives and phenylalanine residuces in dipeptides

Three phenylalanine derivatives, Ac-Phe-NHMe, H-Phe-NHMe, and Ac-Phe-OH, were selected as models of Phe residues situated at the internal, the N-terminal, and the C-terminal positions of peptide chains, respctively. The side-chain conformations of the three compounds were analyzed from the vicnal coupling constants ³J_αβR and ³J_αβS, of their ¹H- nmr spectra measured in various organic sovlent. The two β-protons were unambiguously assined by use of sterospecifically β-monodeuterated phenylalanines. The pro-S β-proton was always situated at lower field than the pro-R one when they were observed separately. The results of a solvent effect on the conformation of the tree compounds demonstrated that the rotamer populations are remarkable sensitive of the three compounds demonstrated that the rotamer populations are remarkably sensitive to solvent polarity and that the tendencies of the solvent effects are quite different from each other. Ac-Phe-OH Showed a trend similar to that of Ac-Phe-OEt reported by early workers. The rotamer populations of other derivatives (Ac-Phe-NMe₂, Ac-Phe-NH₂, Ac-Phe-OBu^t, and Ac-Phe-OBzl) and of Phe residues in some N-acetyl dipeptde esters (Ac-Phe-Gly-OMe, Ac-Phe-Val-OMe, and Ac-Gly-Phe-OMe) were also examined in several sovent, and it was found that substituents of the Phe carboxyl group—amides or esters—determine the tendency of the solvent effect. These results are interesting in the side-chain conformations of Phe residues in peptides and proteins in an environment of low polarity can be disscussed on this experimental basis. Factors responsible for the solvent effect are discussed from (1) a structural comparison of the compunds with various carboxylic substituents, (2) an expriment with cyclohexylalanine derivatives, and (3) the measurement in mixed solvents wiht similar polarity.     

Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase

The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. ¹H–¹⁵N HSQC NMR spectra were obtained of the ¹⁵N-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid.     

Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat

The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13-15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.     

Beta-casomorphins: substitution of phenylalanine with beta-homo phenylalanine increases the mu-type opioid receptor affinity

Two analogues of bovine beta-casomorphin-7 and beta-casomorphin-5 containing a beta-homo phenylalanine in substitution of the phenylalanine in position 3 were synthesised and tested for their mu-opioid receptor affinity. The modification enhanced the mu receptor affinity 5-fold in the case of modified beta-CM-7 and 2-fold for modified beta-CM-5 when compared to the natural peptides.     

Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Characterization of phenylalanine hydroxylase

Iron can be bound to phenylalanine hydroxylase (PAH) in two environments. The assignment of the electron paramagnetic resonance spectrum of PAH to two, overlapping high-spin ferric signals is confirmed by computer simulation. Both environments are shown to be populated in the crude enzyme. Reconstitution of the apoenzyme demonstrated that the two iron environments are not interconvertible. Oxygen consumption during PAH reduction by tetrahydropterin in the absence of phenylalanine but not in its presence explains the different reduction stoichiometries (tetrahydropterin:enzyme) that have been observed.