Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

The iron atom in the nonheme iron monooxygenase phenylalanine hydroxylase is bound on one face by His285, His290, and Glu330. This arrangement of metal ligands is conserved in the other aromatic amino acid hydroxylases, tyrosine hydroxylase and tryptophan hydroxylase. A similar 2-His-1-carboxylate facial triad of two histidines and an acidic residue are the ligands to the iron in other nonheme iron enzymes, including the α-ketoglutarate-dependent hydroxylases and the extradiol dioxygenases. Previous studies of the effects of conservative mutations of the iron ligands in tyrosine hydroxylase established that there is some plasticity in the nature of the ligands and that the three ligands differ in their sensitivity to mutagenesis. To determine the generality of this finding for enzymes containing a 2-His-1-carboxylate facial triad, the His285, His290, and Glu330 in rat phenylalanine hydroxylase were mutated to glutamine, glutamate, and histidine. All of the mutant proteins had low but measurable activities for tyrosine formation. In general, mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All of the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. The H285Q enzyme had the highest activity as tetrahydropterin oxidase at 20% the wild-type value. All of the mutations greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family.     

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.     

The effect of vanadate upon the expression of phenylalanine hydroxylase in streptozotoxin-diabetic rat liver

Induction of diabetes in rats is associated with a significant elevation in the phenylalanine hydroxylating capacity of the liver. This phenomenon reflects an increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine hydroxylase-specific mRNA. These changes can be abolished by insulin-dependent control of diabetes. We show here that the control of diabetes of oral administration of sodium orthovanadate will also nullify the diabetes-related alterations in phenylalanine hydroxylase expression. In addition, diabetes-induced changes in the extent of phosphorylation of phenylalanine hydroxylase are reversed by either or vanadate treatment in vivo. These treatments also abolished the diabetes-related, approx. 30-fold, decrease in glucagon sensitivity of phenylalanine hydroxylation in isolated liver cells.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.     

Estimation of amino acid pairs sensitive to variants in human phenylalanine hydroxylase protein by means of a random approach

In this data-based theoretical analysis, we use a random approach to estimate amino acid pairs in human phenylalanine 4-hydroxylase (PAH) protein in order to determine which amino acid pairs are more sensitive to 187 variants in human PAH protein. The rationale of this study is based on our hypothesis and previous findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs rather than at randomly predictable pairs. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. 94.12% of 187 variants occurred at randomly unpredictable amino acid pairs, which accounted for 71.84% of 451 amino acid pairs in human PAH protein. The chance of a variant occurring is five times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human PAH protein. The results also suggest that the human PAH protein has a natural tendency towards variants.     

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.     

Role of tryptophan hydroxylase phe313 in determining substrate specificity

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.     

Developmental regulation of phenylalanine hydroxylase activity in Drosophila melanogaster

Herein we demonstrate that Drosophila larvae possess a synthetic activity capable of converting phenylalanine to tyrosine. This system is readily extractable and displays many characteristics of phenylalanine hydroxylase systems described in other organisms, the most notable being that a tetrahydropteridine is required for full expression of activity. The level of phenylalanine hydroxylase activity present in the organism varies with the stage of development: from an undetected level of activity at the first larval instar, there is a rapid increase in phenylalanine hydroxylase activity which reaches a peak at the time of puparium formation, after which there is a rapid decrease again to an undetected level.     

Identification of Gln313 and Pro327 as Residues Critical for Substrate Inhibition in Tyrosine Hydroxylase

Abstract: Rat tyrosine hydroxylase was expressed in Escherichia coli . High-level expression was obtained after incubation at 27°C for 18 h. The smallest fragment of tyrosine hydroxylase that gave a soluble active molecule was from Leu¹⁸⁸ to Phe⁴⁵⁶. This fragment corresponds directly to the section of phenylalanine hydroxylase that had previously been shown to be this enzyme's catalytic core region. It has been shown that Glu²⁸⁶ plays a critical role in pterin function in phenylalanine hydroxylase. The corresponding residue in tyrosine hydroxylase (Glu³³²) has no significant role in pterin function. Substitution of a leucine for a proline at position 327 in tyrosine hydroxylase produces a molecule with a K _m for tetrahydrobiopterin 20-fold higher than that of the wild-type molecule, whereas the same substitution at the corresponding residue in phenylalanine hydroxylase (Pro²⁸¹) has no effect on the kinetic constant for the cofactor. This suggests that corresponding residues in phenylalanine hydroxylase and tyrosine hydroxylase can have different roles in pterin function. Substitution of a leucine for a proline at position 281 in phenylalanine hydroxylase increases the K _m for phenylalanine >20-fold over that of the wild-type. Substitution of leucine or alanine for Pro³²⁷ or a glutamic acid for Gln³¹³ in tyrosine hydroxylase eliminates the substrate inhibition shown by wild-type tyrosine hydroxylase.     

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

Iron binding effects on the kinetic stability and unfolding energetics of a thermophilic phenylalanine hydroxylase from Chloroflexus aurantiacus

The effects of non-heme iron binding on the function, structure, and stability of a monomeric phenylalanine hydroxylase from the thermophile Chloroflexus aurantiacus (caPAH) were investigated. Comparative studies on holo (iron-bound) and apo (iron-depleted) caPAH indicated that iron(II) binding does not significantly affect the overall structure of the enzyme. Thermal denaturation studies performed using differential scanning calorimetry showed that the unfolding reaction was kinetically controlled and that holo-caPAH displayed a large increase in thermal stability (approximately 15 °C upshift in the T _m value) compared with the apoenzyme. Analysis using a simple irreversible two-state model also showed a higher kinetic stability for holo-caPAH at optimal growth temperature (denaturing approximately 8 times more slowly than the apo form at 55 °C). Experiments performed in the presence of urea in combination with structure–energetics calculations suggest that iron binding reduces the change in accessible surface area exposed in the unfolding transition state (from approximately 36% to approximately 5% of the total change in accessible surface area) and also the surface involved in water-unsatisfied broken internal contacts (solvation barriers). Additional comparative analyses using phenylalanine hydroxylase from mesophilic and psychrophilic organisms suggest that, in addition to its catalytic role, the non-heme iron serves to enhance the kinetic stability of phenylalanine hydroxylase at the optimal growth temperature of the organism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.     

Spectrum of mutations in Lebanese patients with phenylalanine hydroxylase deficiency

Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2 years 9 months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype–phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region.     

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.     

Relationship between enzymatic activity and oligomerization state of tyrosine hydroxylase

The native form of tyrosine hydroxylase (TH) is a homotetramer which consists of four identical subunits each with an MW of approximately 60 kD. The relationships between the catalytic activity of TH and oligomerization of the enzyme have not yet been characterized. We have investigated, by deletion and/or substitution mutagenesis, the involvement of the leucine zipper (LZ) motifs in the oligomer formation of TH and its relation to catalytic activity. Our results demonstrate that deletion of the carboxyl-terminal LZ (LZ-C) abolishes tetramer formation. Interruption of the other two LZ motifs (LZ-A and LZ-B), located in a central region of the catalytic domain by substitution of Leu to Pro at residues 294 and 301 or 386 and 393 has no effect on the tetramer formation of TH. However, the interruption of LZ-A and LZ-B abolishes TH enzymatic activity. The substitution of Leu residues 188 and 190 with Pro at the regulatory domain of TH reduces enzymatic TH activity without affecting tetramer formation. Thus, LZ-C is required for tetramer formation, while LZ-A and LZ-B seem to be involved in the catalytic activity without affecting the tetramer formation of TH.     

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.     

Glu434 is an important amino acid residue for the activity,structure and stability of tyrosine hydroxylase of the silkworm,Bombyx mori

In this study, silkworm (Bombyx mori) tyrosine hydroxylase was expressed in Escherichia coli. The enzymatic characteristics of the recombinant wild silkworm tyrosine hydroxylase were similar to that of native silkworm tyrosine hydroxylase (BmTH). We investigated the role of the amino acid residue Glu434 of BmTH using site-directed mutagenesis. The activity of the E434A mutant was approximately 35.6 percent of that exhibited by BmTH. Furthermore, the mutation dramatically reduced its substrate affinity for tetrahydrobiopterin and decreased its activation by Fe²⁺. The E434A mutation impaired the conformational structure of BmTH, resulting in a partially unfolded state with more hydrophobic exposure, a tendency to aggregate and structural instability during environmental stresses. This mutation did not significantly affect a three-step transitional folding process involving two intermediate states in GdnHCl. However, it did affect the structural compactness of the folding intermediates. The results suggest that the Glu434 residue is an important determinant of the activity, stability and conformational structure of BmTH.     

Simultaneous measurement of phenylalanine and tyrosine in phenylketonuric plasma and dried blood by high-performance liquid chromatography

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with alpha-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm x 4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.