Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

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.     

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.     

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 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.     

A new enzyme, NADPH-dihydropteridine reductase in bovine liver.

An enzyme designated as NADPH-dihydropteridine reductase was found in the extract of bovine liver and partially purified. In contrast to NADH-dpendent dihydropteridine reductase [EC 1.6.99.7], the enzyme catalyzes the reduction of quinonid-dihydropterin to tetrahydropterin in the presence of NADPH. The two enzymes were separated by column chromatography on DEAE-sephadex. Tyrosine formation in the phenylalanine hydroxylation system was also stimulated by NADPH-dihydropteridine reductase. The existence of these two dihydropteridine reductases suggests that the tetrahydro from ofpteridine cofactor may be regenerated in two different ways in vivo.     

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.     

The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum

The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.     

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.     

The AP-1 repressor, JDP2, is a bona fide substrate for the c-Jun N-terminal kinase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.     

Evidence for the formation of the 4a-carbinolamine during the tyrosine-dependent oxidation of tetrahydrobiopterin by rat liver phenylalanine hydroxylase

In the presence of phenylalanine and molecular oxygen, activated phenylalanine hydroxylase catalyzes the oxidation of tetrahydrobiopterin. The oxidation of this tetrahydropterin cofactor also proceeds if the substrate, phenylalanine, is replaced by its product, tyrosine, in the initial reaction mixture. These two reactions have been defined as coupled and uncoupled, respectively, because in the former reaction 1 mol of phenylalanine is hydroxylated for every mole of tetrahydrobiopterin oxidized, whereas in the latter reaction there is no net hydroxylation of tyrosine during the oxidation of the tetrahydropterin. During the course of the coupled oxidation of tetrahydrobiopterin, a pterin 4a-carbinolamine intermediate can be detected by ultraviolet spectroscopy (Kaufman, S. (1976) in Iron and Copper Proteins (Yasunobu, K. T., Mower, H. F., and Hayaishi, O., eds) pp. 91-102, Plenum Publishing Corp., New York). Dix and Benkovic (Dix, T. A., and Benkovic, S. J. (1985) Biochemistry 24, 5839-5846) have postulated that the formation of this intermediate only occurs when the oxidation of the tetrahydropteridine is tightly coupled to the concomitant hydroxylation of the aromatic amino acid. However, during the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase, we have detected the formation of a spectral intermediate with ultraviolet absorbance that is essentially identical to that of the carbinolamine. Furthermore, this absorbance can be eliminated by the addition of 4a-carbinolamine dehydratase, an enzyme which catalyzes the dehydration of the 4a-carbinolamine. Quantitation of this intermediate suggests that there are two pathways for the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase because only about 0.3 mol of the intermediate is formed per mol of the cofactor oxidized.     

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.     

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.     

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     

An additional substrate binding site in a bacterial phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.     

Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality?

Background

Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine) and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function) in a relatively large sample of schizophrenia patients and healthy controls.

Methods

We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls.

Results

Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001) and phenylalanine: tyrosine ratio (p<0.0001) but tyrosine did not differ between the two groups (p = 0.596).

Conclusions

Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches.     

Expression of one isoform of GTP cyclohydrolase I coincides with the larval black markings of the swallowtail butterfly, Papilio xuthus

The larva of the swallowtail butterfly Papilio xuthus changes its body markings during the fourth ecdysis. We found that stage-specific cuticular black markings are mainly regulated by co-localization of two melanin synthesis enzymes; tyrosine hydroxylase (TH) and dopa decarboxylase (DDC). TH converts tyrosine to dihydroxyphenylalanine (dopa), and tyrosine itself is converted from phenylalanine by phenylalanine hydroxylase (PAH). Guanosine triphosphate cyclohydrolase I (GTPCHI) is essential for the synthesis of tetrahydrobiopterin (BH4) that is a cofactor of TH and PAH. In this report, we found that a GTPCHI inhibitor prevents pigmentation in cultured integuments, suggesting that the GTPCHI activity is also involved in cuticle pigmentation. We have cloned GTPCHI and PAH cDNAs from P. xuthus and investigated their spatial expression patterns in epidermis by whole-mount in situ hybridization. There are two isoforms of GTPCHI in larval epidermis (GTPCHIa and GTPCHIb). GTPCHIa is expressed at the black markings of the subsequent instar, similar to TH, whereas GTPCHIb is expressed uniformly, similar to PAH. This suggests that the region-specific expression of GTPCHIa supplies sufficient BH(4) reinforcing the TH activity in black marking area. Our results imply that larval markings are regulated by not only melanin synthesis enzymes but also the cofactor supplying enzyme.     

An oxygraphic method for determining kinetic properties and catalytic mechanism of aromatic amino acid hydroxylases

We have developed a simple and versatile oxygraphic assay procedure that can be used for determination of kinetic constants and enzyme reaction mechanisms of wild-type and mutant aromatic amino acid hydroxylases. The oxygen concentration and rate of oxygen consumption were measured continuously throughout the enzyme reaction, while aliquots of the reaction mixture were removed at regular intervals for measurement of other substrates and products. Using (6R)-tetrahydrobiopterin as electron donor in the phenylalanine hydroxylase (PAH) reaction, a stable stoichiometry of 1:1 was obtained between the amount of oxygen consumed and the tyrosine formation. In comparison, low and variable coupling efficiency values between oxygen consumption and tyrosine formation were found using the parent unsubstituted tetrahydropterin. The application of this assay procedure to study mechanisms of disease-associated mutations was also demonstrated. Thus, the phenylketonuria-associated PAH mutant R158Q had a coupling efficiency of about 80%, compared to the wild-type enzyme under similar conditions. Furthermore, the amount of H(2)O(2) produced in the reaction catalyzed by R158Q PAH was about four times higher than the amount produced by the wild-type PAH, demonstrating a possible pathogenetic mechanism of the mutant enzyme.     

Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.