Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (k_cat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K_M values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the K_M values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K_M value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k_cat/K_Phe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH₂ 7,8-dihydro-l-biopterin - BH₄ (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH₄ 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH₄ tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type     

Pteridine-requiring dihydroorotate hydroxylase from Crithidiafasciculata

Dihydroorotic acid is converted to orotic acid in $\text{Crithidia}$ by hydroxylation and subsequent dehydration. The hydroxylase is soluble, stable to acid (pH 4.0), destroyed by alkali (pH 11) and by heat (55° for 3 min.). Activity is rapidly lost upon standing at 4° and upon freezing. Its activity is optimum at pH 7.4. Its isoelectric point is 6.2. It has an absolute dependence on O₂ and a reduced pteridine. Pteridine reductases are present in cell extracts which, in the presence of NADH, permits the efficient use of biopterin (the oxidized form of the naturally occurring pteridine in this organism) as a cofactor for the hydroxylase.     

Biopterin cofactor and monoamine-synthesizing monooxygenases

The activities of three pterin-requiring monooxygenases, phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, are regulated by the level of the pterin cofactor, $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$, which is synthesized from guanosine triphosphate (GTP). Since tyrosine hydroxylase or tryptophan hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, norepinephrine and epinephrine) or serotonin in monoaminergic neurons, biosynthesis of tetrahydrobiopterin from GTP may also regulate the tissue level of monoamine transmitters. Recent evidences indicate that biosynthesis of tetrahydrobiopterin and that of biogenic monoamines may be regulated each other.     

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.     

The interaction of aromatic amino acids with rat liver phenylalanine hydroxylase

We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Inhibition of phenylalanine hydroxylase by p-chlorophenylalanine; dependence on cofactor structure

The reported discrepancy between the in vitro and in vivo properties of p-chlorophenylalanine as an inhibitor of phenylalanine hydroxylase (E.C.1.14. 3.1) was investigated. It was demonstrated that the lack of inhibition, in vitro, was not due to (1) non-physiological pH or temperature of the in vitro assay system, (2) inhibition by m-chlorotyrosine, a product of the enzymatic hydroxylation of p-chlorophenylalanine, or (3) a slow irreversible reaction of p-chlorophenylalanine with enzyme. However, when the inhibitory properties of p-chlorophenylalanine were determined using the natural cofactor, tetrahydrobiopterin, instead of the pseudocofactor 6,7-dimethyltetrahydropterin, which had been utilized in the reported in vitro studies, it was shown that p-chlorophenylalanine is a potent inhibitor of the enzymatic hydroxylation of phenylalanine. The apparent K_i is 0.03mM with tetrahydobiopterin as cofactor, compared to 1.5mM with 6.7-dimethyltetrahydropterin. The dependence of the inhibitory properties of an aromatic amino acid analog on the structure of the cofactor may be a general phenomenon with all tetrahydrobiopterin dependent aromatic amino acid hydroxylases.     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

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.     

A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.     

Methylfolate modulates potassium evoked neuro-secretion: Evidence for a role at the pteridine cofactor level of tyrosine 3-hydroxylase

We have previously shown that 5-methyltetrahydrofolate influences neuro-secretion. The present study more precisely characterises the processes involved and considers one probable site of action. Focusing on the tyrosine-noradrenalin axis in cerebellum we showed 5-methyltetrahydrofolate causes a significant reduction in the apparent K⁺ evoked secretion of noradrenalin to only 12.9% of control release. Evidence supports the idea that this could actually be due to increased synthesis leading to; depletion of reserves, possibly through leakage, exocytotic inhibition via activation of presynaptic receptors or end product inhibition by noradrenalin at the pteridine cofactor level of tyrosine hydroxylase: a) concomitant decreased measurement of perfusate and intracellular tyrosine with released noradrenalin following 5-methyltetrahydrofolate treatment supports the idea of increased transmitter turn over; b) kinetic studies indicate that at saturating concentrations of tyrosine and in the presence of an inhibitor of L-DOPA decarboxylase, 5-methyltetrahydrofolate partially duplicates the rate limiting behaviour of a synthetic pteridine cofactor — DL,2-amino-4-hydroxy-6,7,dimethyltetrahydropteridine. We debate whether, in vivo, CSF 5-methyltetrahydrofolate might interact at the tetrahydrobiopterin cofactor level of tyrosine hydroxylase and other aromatic amino-acid hydroxylases.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

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.     

Tyrosine hydroxylase of sheep brain: some catalytic and chemical properties of the detergent-solubilized, partially purified enzyme

Abstract– Detergent-solubilized tyrosine hydroxylase from the caudate nucleus of the sheep was purified 3-fold by affinity chromatography on 3-iodotyrosine modified agarose. Supplementation of the standard assay with 1 mM Fe²⁺ resulted in only slight stimulation of the enzymic activity. The enzyme was, however, markedly inhibited by certain complexing agents specific for either Fe²⁺ or Fe³⁺. Double reciprocal plots of the kinetic data for a representative complexing agent, bathophenanthroline, showed the inhibition to be competitive with O₂ (apparent K_m 0.15 mM) and noncompetitive with either l -tyrosine or the synthetic tetrahydropterin cofactor DMPH₄ (apparent K_m's 0.12 and 0.29 mM, respectively). The combined inhibition and kinetics studies strongly suggest that brain tyrosine hydroxylase is an iron enzyme. Furthermore, the prosthetic iron very likely participates directly in catalytic function, presumably by binding molecular oxygen. The activity of ammonium sulphate-precipitated enzyme was found to be stimulated 2-fold by Fe²⁺, catalase or peroxidase, while untreated enzyme was markedly less affected by these agents. Since the only ostensible difference between the two preparations was the extensive aggregation present in the former case, the change in physical state evoked by ammonium sulphate precipitation appeared to be somehow related to this peculiar property of the enzyme.     

The identification of MYO-inositol:NAD(P)+ oxidoreductase in mammalian brain.

$\text{myo}$-Inositol:NAD(P)⁺ oxidoreductase ($\text{myo}$-inositol oxidoreductase) has been identified in bovine brain. This enzyme elutes from DEAE cellulose with 0.3 M KCl in 50 mM Tris buffer, pH 7.5. Using NADH as cofactor $\text{myo}$-inosose-2 is reduced selectively to $\text{myo}$-inositol. With NADPH the enzyme forms both $\text{myo}$-inositol and $\text{scyllo}$-inositol, however, at a lower rate. The enzyme was chromatographed on G-100 Sephadex and found to have an apparent molecular weight of 74,000. This enzyme differs in DEAE binding, molecular weight and cofactor specificity from the previously described $\text{scyllo}$-inositol oxidoreductase which utilizes NADPH exclusively to produce 3 fold more $\text{scyllo}$-inositol than $\text{myo}$-inositol.     

Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P-450 dependent monooxygenase.

An end-product indole alkaloid, catharanthine, inhibits a membrane-bound cytochrome P-450 dependent monooxygenase of the higher plant, $\text{Vinca rosea}$. Kinetic analysis revealed the alkaloid to be a reversible, linear, non-competitive inhibitor (K_i=1 mM) with respect to its substrates, geraniol and NADPH. Comparable inhibition of the solubilized monooxygenase by catharanthine tends to exclude a mechanism based upon disruption of membrane organization. On the basis of its inhibition of solubilized hydroxylase in the presence and absence of sodium cholate, it is also unlikely that catharanthine competes for putative phospholipid binding site(s). Two additional end-product alkaloids, vinblastine and vindoline were less inhibitory. Since the hydroxylase catalyzes one of the first committed steps in the biosynthesis of indole alkaloids, these observations suggest feedback control of the pathway by catharanthine.     

Determination of phenylalanine hydroxylase activity by liquid chromatography/electrochemistry

An assay method is presented for the determination of phenylalanine hydroxylase activity in biological samples. The procedure is rapid and requires little sample. Multiple components of the enzyme system are determined and therefore serve as internal checks of the assay system. Liquid chromatography/electrochemistry is employed to follow the oxidation of the tetrahydropterin cofactor to the dihydropterin and to follow the formation of tyrosine. The KM and Vmax values of both phenylalanine and 6-methyl-5,6,7,8-tetrahydropterin were determined for mouse liver phenylalanine hydroxylase. Determination of the stoichiometry of the reaction showed that 1 mol of dihydropterin and 1 mol of tyrosine are formed per mole of tetrahydropterin that is oxidized. The reaction rate was linear for several minutes and over a wide range of enzyme (protein) concentrations.     

Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates

The biosynthetic pathway of tetrahydrobiopterin (BH₄) from dihydroneopterin triphosphate (NH₂P₃) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH₂P₃ was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH₄ to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH₄ biosynthesis under normal $\text{in vivo}$ conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH₂P₃ may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH₄ by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH₄ biosynthesis $\text{in vivo}$.     

Reaction of rat liver phenylalanine hydroxylase with fatty acid hydroperoxides. Characterization and mechanism

Rat liver phenylalanine hydroxylase must be in a reduced form to be catalytically active (Marota, J.J. A., and Shiman, R. (1984) Biochemistry 23, 1303-1311). In this communication we show that a fatty acid hydroperoxide, 13-hydroperoxylinoleic acid (LOOH), can efficiently oxidize the reduced enzyme. In the process, the hydroperoxide is decomposed, oxygen consumed, and hydrogen peroxide formed. Enzyme reduction by the tetrahydropterin cofactor and reoxidation by LOOH can occur as two single steps or, when the enzyme concentration is low compared to that of the substrates, as part of a catalytic cycle. In this latter case, phenylalanine hydroxylase is a hydroperoxide-dependent tetrahydropterin oxidase. The reaction requires 1.0 mol of O2, 1.0 mol of tetrahydropterin, and 0.5 mol of LOOH to yield 1.0 mol of quinonoid dihydropterin, 0.4 mol of H2O2, and fatty acid products. Thus far, the catalytic and single-step reactions appear the same in all properties, consistent with the steady-state reaction following a ping-pong mechanism. Phenylalanine hydroxylase is an excellent catalyst for this reaction: the turnover number with LOOH is slightly greater than with phenylalanine; the Km(app) for LOOH is 11 +/- 4 microM; and the kcat/Km ratio for LOOH is about 25 times greater than for phenylalanine. LOOH and phenylalanine appear to react at different sites on phenylalanine appear to react at different sites on phenylalanine hydroxylase, and the reaction of LOOH is inhibited only slightly by phenylalanine and not at all by 5-deaza-6-methyltetrahydropterin, a competitive inhibitor of phenylalanine hydroxylation. The reaction of LOOH with phenylalanine hydroxylase strongly resembles the nonenzymatic reaction of LOOH with hematin, implying similar mechanisms for the two reactions and implicating the enzyme's non-heme iron as both the site of reaction of LOOH and of electron transfer during oxidation and reduction. The formation of hydrogen peroxide during a reaction of phenylalanine hydroxylase is unusual. Indirect evidence indicates a reduced oxygen species, formed on the enzyme during the reduction step, is (partially) released as H2O2 when the hydroperoxide reacts.