Tyrosine Hydroxylation Research Articles

Intrinsic deuterium isotope effects on benzylic hydroxylation by tyrosine hydroxylase.

Tyrosine hydroxylase (TyrH) is a mononuclear, non-heme iron monooxygenase that catalyzes the pterin-dependent hydroxylation of tyrosine to dihydroxyphenylalanine. When 4-methylphenylalanine is used as a substrate for TyrH, 4-hydroxymethylphenylalanine is one of the amino acid products. To examine the mechanism of benzylic hydroxylation, the products and their isotopic compositions were determined with 4-methylphenylalanines containing a mono-, di-, or trideuterated methyl group as substrates. Intrinsic primary and secondary deuterium isotope effects for benzylic hydroxylation of 9.6 +/- 0.9 and 1.21 +/- 0.08, respectively, were derived from the data. The magnitudes of these isotope effects are consistent with quantum mechanical tunneling of the hydrogen. The similarity of the effects to those seen for benzylic hydroxylation by other enzymes supports a mechanism where a high valence iron-oxo species, Fe(IV)=O, is the hydroxylating intermediate.

Diphenol activation of the monophenolase and diphenolase activities of field bean (Dolichos lablab) polyphenol oxidase.

This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean (Dolichos lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which decreased with increasing catechol concentration and increased with increasing ferulic acid concentration. The activation constant K(a) of catechol for ferulic acid hydroxylation reaction was 5 mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and 2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase, could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase (Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine, and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs immediately with no lag period and does not occur without the addition of diphenol. The kinetic parameters for caffeic acid (K(m) = 0.08 mM, V(max) = 32440 u/mg) in the presence of catechol and the activation constant K(a) of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag period for this reaction indicates that the diphenol mechanism of diphenolase activation differs from the way in which the same o-diphenols activate the monophenolase activity.

Supersensitivity of 5-HT1A autoreceptors and alpha2-adrenoceptors regulating monoamine synthesis in the brain of morphine-dependent rats.

The sensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) modulating brain monoamine synthesis was investigated in rats during morphine treatment and after naloxone-precipitated withdrawal. The accumulation of 5-hydroxytryptophan (5-HTP) and 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition was used as a measure of the rate of tryptophan and tyrosine hydroxylation in vivo. Acute morphine (3-100 mg/kg, 1 h) increased the synthesis of 5-HTP/5-HT in various brain regions (15%-35%) and that of DOPA/dopamine (DA) in striatum (28%-63%), but decreased the synthesis of DOPA/noradrenaline (NA) in hippocampus and cortex (20%-33%). Naloxone (2-60 mg/kg, 1 h) did not alter the synthesis of 5-HTP or DOPA in brain. Tolerance to the inhibitory effect of morphine on DOPA/NA synthesis and a sensitization to its stimulatory effects on DOPA/DA and 5-HTP/5-HT synthesis were observed after chronic morphine and/or in morphine-withdrawn rats. In morphine-dependent rats (tolerant and withdrawn states) the inhibitory effects of the 5-HT1A agonists 8-OH-DPAT and buspirone (0.1 mg/kg, 1 h), and that of the alpha2-adrenoceptor agonist clonidine (0.1 mg/kg, 1 h), on the synthesis of 5-HTP/5-HT were potentiated (25%-50%). Moreover, the effect of 8-OH-DPAT was antagonized by WAY 100135, a selective 5-HT1A antagonist. In morphine-dependent rats (tolerant state), the inhibitory effects of clonidine on the synthesis of DOPA/NA (hippocampus, hypothalamus) and DOPA/DA (striatum) also were potentiated (35%-55%). In summary, we conclude that morphine addiction is associated with supersensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) that modulate the synthesis of monoamines in brain.

Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus

A heat-labile protein has been identified in fruit bodies of the edible mushroom, Agaricus bisporus, which protects Raji cells (a human lymphoma cell line) against H 2O 2-induced oxidative damage to cellular DNA. This protein has been purified following salt fractionation, combined with ion-exchange, hydrophobic interaction and adsorption chromatography. Based on catalytic and electrophoretic properties, and inhibition studies using tropolone, the protein was identified as tyrosinase. The genoprotective effect of A. bisporus tyrosinase, determined using the single-cell gel electrophoresis (“Comet”) assay, has been shown to be dependent upon the enzymic hydroxylation of tyrosine to L-DOPA and subsequent conversion of this metabolite to dopaquinone. The possible role of dopaquinone, and other L-DOPA oxidation products, in enhancing the cellular antioxidant defence mechanisms is discussed.

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol).

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

Aminoacyl-S-enzyme intermediates in beta-hydroxylations and alpha,beta-desaturations of amino acids in peptide antibiotics.

Many of the alpha-amino acids found in proteins are shunted into microbial secondary metabolism to form peptide antibiotics by specific oxidation, including hydroxylation, at the beta carbon. Examples for the enzymatic hydroxylation of tyrosine and histidine and for desaturation of proline during covalent attachment as aminoacyl-S-pantetheinyl enzyme intermediates suggest a general strategy in peptide antibiotic biosynthesis.

Manganese mimics the action of 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in rat striatal tissue slices

Manganese and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are known to induce neurological pathologies similar to that of parkinsonism. Previous studies performed in rat striatal slices have shown that MPTP and related compounds inhibit tyrosine hydroxylation, a rate-limiting step of dopamine biosynthesis. Here, we reported that manganese inhibited tyrosine hydroxylation in rat striatal slices. In addition, manganese caused increase in the levels of lactate indicating that aerobic glycolysis was inhibited in striatal slices. This inhibition was unique to manganese since other divalent cations, such as magnesium and zinc, did not increase lactate concentrations. These results suggest that the mechanisms by which manganese produces dysfunction of the nervous system are similar to those of MPTP.

Transforming growth factor β1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation

Transforming growth factor-β1 (TGFβ1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by α-melanocyte stimulating hormone (αMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFβ1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFβ1 (10 −10 M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFβ1 and αMSH on melanosome number, volume density and maturation degree. αMSH (10 −7 M, 48 h) increased 7-fold melanosome volume density, whereas TGFβ1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or αMSH-treated cells. Moreover, TGFβ1 blocked the αMSH-elicited increase in the number of melanosomes. Control and αMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFβ1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with αMSH and TGFβ1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFβ1 and with its hypopigmenting effect.

Crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.

Abnormal presynaptic catecholamine regulation in a hyperactive SNAP-25-deficient mouse mutant

The consequences of a reduction in the presynaptic protein, SNAP-25, were investigated to determine the neurochemical basis of the marked hyperlocomotor activity in coloboma (Cm/+) mice. SNAP-25 is part of the minimal presynaptic machinery necessary for exocytotic neurotransmitter release. Reserpine treatment was used to deplete vesicular stores of catecholamines. Coloboma mice were more sensitive to the effects of reserpine than control mice. However, presynaptic regulation of dopamine (DA) release, as assessed by low-dose apomorphine challenge, was intact. There were region-specific reductions in in vivo tyrosine hydroxylation and the DA metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum and nucleus accumbens of Cm/+ mice. While hyperactivity is often associated with changes in DA concentration, norepinephrine (NE) concentration was significantly increased in the striatum and nucleus accumbens of the hyperactive mutant. The increase in NE may regulate the hyperactivity in these mice, as suggested by current hypotheses of the mechanisms underlying attention-deficit hyperactivity disorder (ADHD) and Tourette's syndrome.

Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B(0).

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the "serine analogues" of pneumocandins B(0) and B(5) and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B(0) and E(0), whereas pneumocandins C(0) and D(0) decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit alpha-ketoglutarate-dependent dioxygenases, affected the pneumocandin B(0) titer and altered the levels of pneumocandins B(1), B(2), B(5), B(6), and E(0), analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns.

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid–base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR–Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells.

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.

O–O bond splitting mechanism in cytochrome oxidase

Hybrid density functional theory (DFT) calculations have been used to investigate different mechanisms for O–O bond splitting in cytochrome oxidase. It is shown that the requirement for a low activation barrier for the O–O bond splitting is that two protons, apart from the tyrosine hydroxyl proton, are available at the binuclear center. A mechanism is suggested for the transformation from a species with a molecularly coordinated O 2, to an O–O cleaved species with an oxo-ferryl group. The mechanism has a calculated activation barrier in reasonable agreement with experimental estimates, and the overall reaction is close to thermoneutral, in line with the requirement that the energy wasted as heat should be minimized. The rate limiting step in the mechanism occurs at the initial Fe–O 2 intermediate, consistent with experimental observations that the decay of the oxy intermediate parallels the increase of the oxo product. The formation of a radical at the cross-linked tyrosine–histidine structure is a possible source for one of the electrons required in the bond cleavage process. Possible sources for the two protons are discussed, including a suggested key role for the hydroxyl group on the farnesyl side chain of heme a 3.

Probing acrylamide alkylation sites in cysteine-free proteins by matrix-assisted laser desorption/ionisation time-of-flight.

It is recognised that gel-separated proteins can experience a frequent modification provoked by the interaction of unpolymerized acrylamide monomers with the thiol group of cysteine to form a beta-cysteinyl-S-propionamide adduct. Other groups which have been implicated in this reaction include the hydroxyl group of tyrosine, the straightepsilon-amino group of lysine, and the free N-terminus. In a series of recent publications it has been demonstrated that at pH approximately 9.5 and in the presence of cysteine, none of these groups experienced measurable interaction with acrylamide monomers. To emphasise this conclusion we have used matrix-assisted laser desorption/ionisation with a reflectron time-of-flight mass spectrometer to examine a number of cysteine-free proteins incubated for various intervals with 30 mM acrylamide monomers at pH 9.5. These high resolution data suggest that, for short incubation times (>/=1 hour) and in the absence of cysteine, the straightepsilon-NH(2) group of lysine is the likely adduction site of acrylamide. Longer incubation times (>/=24 hours) with acrylamide monomers rendered the role of Cys as the favourite alkylation site less evident.

X-ray structure of azide-bound fully oxidized cytochrome c oxidase from bovine heart at 2.9 A resolution.

Two azide ions were identified, one between the Fe and Cu atoms in the O(2)-reduction site and the other at the transmembrane surface of the enzyme, in the crystal structure of the azide-bound form of bovine heart cytochrome c oxidase at 2.9 A resolution. Two geometries, a mu-1,3 type geometry between the Fe and Cu atoms and a terminal geometry on the Fe atom, are equally possible for an azide ion in the O(2)--reduction site. The other azide molecule was hydrogen bonded to an amide group of an asparagine and a hydroxyl group of tyrosine in a mu-1,1 type geometry. The antisymmetric infrared bands arising from these azide ions, which show essentially identical intensity [Yoshikawa & Caughey (1992), J. Biol. Chem. 267, 9757-9766], strongly suggest terminal binding of the azide to Fe. The electron density of all three imidazole ligands to Cu(B) was clearly seen in the electron-density map of the azide-bound form of bovine heart enzyme, in contrast to the crystal structure of the azide-bound form of the bacterial enzyme [Iwata et al. (1995), Nature (London), 376, 660-669], which lacks one of the three imidazole ligands to Cu(B).

Role of the Carbonate Radical Anion in Tyrosine Nitration and Hydroxylation by Peroxynitrite

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate–carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3,3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate–carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate–carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.

Intracranial self-stimulation increases differentially in vivo hydroxylation of tyrosine but similarly in vivo hydroxylation of tryptophan in rat medial prefrontal cortex, nucleus accumbens and striatum

We have examined using microdialysis the effect of intracranial self-stimulation (ICSS) on the in vivo hydroxylation rate of tyrosine and tryptophan in the medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and striatum (STR). A decarboxylase inhibitor NSD-1015 was included in the perfusate, which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo hydroxylation level of tyrosine and tryptophan. When rats were exposed to 1 h of ICSS at the medial forebrain bundle (MFB), their extracellular levels of DOPA significantly increased in the mPFC, NAC and STR, but with a different magnitude and time course. The same stimulation produced a delayed increase in extracellular 5-HTP, compared to DOPA, in these brain regions. The profile of 5-HTP response demonstrated no apparent difference among the regions. These findings indicate that ICSS of the MFB can increase differentially the in vivo hydroxylation of tyrosine but similarly the in vivo hydroxylation of tryptophan in the mPFC, NAC and STR.

Steady-state and Transient Kinetics of Escherichia coli Nitric-oxide Dioxygenase (Flavohemoglobin): THE B10 TYROSINE HYDROXYL IS ESSENTIAL FOR DIOXYGEN BINDING AND CATALYSIS

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.

Mutation of Serine 395 of Tyrosine Hydroxylase Decouples Oxygen−Oxygen Bond Cleavage and Tyrosine Hydroxylation

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.