In vitro expression analysis of R68G and R68S mutations in phenylalanine hydroxylase gene

Phenylketonuria (PKU), an autosomal recessive disorder caused be a deficiency of hepatic phenylalanine hydroxylase (PAH), is clinically very heterogeneous. At the molecular level, more than 400 mutations in the PAH gene are known to date, which in different genotype combinations could account for biochemical and clinical variability of symptoms. In vitro expression studies on R68G and R68S mutations causing mild phenylketonuria are presented.     

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.     

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.     

Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca²⁺ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca²⁺ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.     

Allosteric regulation of phenylalanine hydroxylase

The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.     

The mechanism of phenylalanine hydroxylase

The site of oxygen binding during phenylalanine hydroxylase (PAH)-catalyzed turnover of phenylalanine to tyrosine has been tentatively identified as the 4a position of the tetrahydropterin cofactor, based on the spectral characteristics of an intermediate generated from both 6-methyltetrahydropterin and tetrahydrobiopterin during turnover. The rates of appearance of the intermediate and tyrosine are equal. Both rates exhibit the same dependence on enzyme concentration. PAH also requires 1.0 iron per 50,000-dalton subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated. Apoenzyme can be reactivated by addition of Fe(II) aerobically or Fe(III) anaerobically and can be repurified to give apparently native protein. Evidence from electron paramagnetic resonance implicates the presence of high spin (5/2) Fe(III). As a working hypothesis we postulate that a key complex at the active site may be one containing iron in close proximity to a 4a-peroxytetrahydropterin.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

Haplotype distribution and molecular defects at the phenylalanine hydroxylase locus in Italy

Summary In order to investigate the molecular basis of phenylketonuria (PKU) in Italy, we characterized the RFLP haplotypes at the phenylalanine hydroxylase gene in 38 unrelated Italian PKU families. The distribution of haplotypes associated with PKU alleles differs from that of other European populations. In particular, haplotypes 1 and 6 are present in 39.7% and 17.6% of the PKU chromosomes, whereas the frequencies of haplotypes 2 and 3 are 5.9% and 2.9%, respectively. The characterization of PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides shows that 1 out of 2 haplotypes 3 carries the splicing mutation and that 2 out of 4 haplotypes 2 carry the missense mutation associated with these haplotypes in North European populations. Our results indicate that the two molecular defects most frequent in Northern Europe represent a minority of PKU mutations in Italy.     

p-Chlorphenylalanine effect on phenylalanine hydroxylase in hepatoma cells in culture.

We have investigated the p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase activity in cultured hepatoma cells. The similarity of the effect of p-chlorophenylalanine on phenylalanine hydroxylase in the hepatoma cells and that reported from studies in vivo indicates that the loss of phenylalanine hydroxylase activity is due to a direct interaction of the amino acid analogue with the liver. We can find no evidence that the loss of phenylalanine hydroxylase activity is due to: a direct inactivation of the hydroxylase by p-chlorophenylalanine or an inhibitor produced by p-chlorophenylalanine treatment; an effect similar to that of p-fluorophenylalanine; or leakage of enzyme from the cells during p-chlorophenylalanine treatment. The data presented indicate: (a) the p-chlorophenylalanine effect is rather specific for phenylalanine hydroxylase; (b) following p-chlorophenylalanine removal, new protein synthesis is necessary for restoration of the hydroxylase activity; (c) the rate of loss of phenylalanine hydroxylase activity after the addition of p-chlorophenylalanine is much faster than the rate of restoration of the hydroxylase activity after removal of p-chlorophenylalanine; (d) even in the presence of p-chlorophenylalanine, hydrocortisone greatly stimulates the hydroxylase activity; (e) the cell density-dependent increase of phenylalanine hydroxylase activity is blocked by p-chlorophenylalanine. A discussion of the possible mechanisms of p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low leanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low levels of phenylalanine hydroxylase activity, a new procedure, based on isotope dilution, was developed for isolating the tyrosine formed during the enzymatic reaction.     

Urea-induced denaturation of human phenylalanine hydroxylase

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of the fusion partner, the effects of increasing urea concentrations on enzyme activity, aggregation, unfolding, and refolding were examined. At pH 7.50, purified human phenylalanine hydroxylase is transiently activated in the presence of 0-4 M urea but slowly inactivated at higher denaturant concentrations. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phenylalanine (L-Phe) shifts the transition midpoint of the first transition from 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant differences in dDeltaG(D)/d[urea] (m(D) values) were observed for the first denaturation transition. In the absence of substrate, a high rate of non-covalent aggregation was observed for the enzyme in the presence of 1-4 M urea. All three tryptophan residues in the enzyme (Trp-120, Trp-187, and Trp-326) were mutated to phenylalanine, either as single mutations or in combination, in order to identify the residues involved in the spectroscopic transitions. A gradual dissociation of the native tetrameric enzyme to increasingly denatured dimeric and monomeric forms was demonstrated by size exclusion chromatography in the presence of denaturants.     

The mechanism of the irreversible inhibition ofrat liver phenylalanine hydroxylase due to treatment with p-chlorophenylalanine. The lack of effect on turnover of phenylalanine hydroxylase.

The administration of a single dose of p-chlorophenylalanine (360 mg/kg) to rats leads to the irreversible loss of 90% of hepatic phenylalanine hydroxylase activity after 24 h. This loss of activity is not the result of either an alteration in the overall structure of the enzyme, as determined by its antigenicity, or in the total immunologically reactive protein in the liver, as tested with a specific antiserum prepared against native phenylalanine hydroxylase. Neither the rate of synthesis nor the rate of degradation of phenylalanine hydroxylase is changed by p-chlorophenylalanine (pClPhe) treatment. The half-life for the enzyme is about 2 days in control and in pClPhe-treated rats. In addition, there is no detectable incorporation of pClPhe into the phenylalanine hydroxylase molecule itself.     

Sequence variation at the phenylalanine hydroxylase gene in the British Isles.

Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations.     

The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity.

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.     

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.     

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria

Summary Eight polymorphic restriction enzyme sites at the phenylalanine hydroxylase (PAH) locus were analyzed from the parental chromosomes in 33 Danish nuclear families with at least one phenylketonuric (PKU) child. Determination of haplotypes of 66 normal chromosomes and 66 chromosomes bearing mutant allele (S) demonstrated that there are at least two haplotypes which occur predominantly on PKU chromosomes and rarely otherwise. Overall, the relative frequencies of the various haplotypes are significantly different on PKU-and normal-allele bearing chromosomes, even though there is no predominantly occurring unique haplotype which can characterize the PKU chromosomes. In addition, no significant association (linkage disequilibrium) between any single polymorphic site and the mutant allele (s) was observed. The results suggest that either the phenylketonuric mutation was very ancient so that the polymorphic sites and the mutation have reached linkage equilibrium or the mutant allele (s) are the results of multiple mutations in the phenylalanine hydroxylase gene in man. Furthermore, a crude relationship between standardized linkage disequilibria and physical map distances of the polymorphic sites indicates that there is no apparent recombination hot-spot in the human phenylalanine hydroxylase gene, since the recombination rate within the locus apears to be uniform and likely to be occurring at a rate similar to that within the HLA gene cluster. The limitations of this later analysis are discussed in view of the sampling errors of disequilibrium measure used, and the potential untility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.