Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.

The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.     

Inhibition of GTP cyclohydrolase I by pterins

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

A review of recent advances on the regulation of pigmentation in the human epidermis.

It has been recognised that the active transport of L-phenylalanine and its autocrine turnover to L-tyrosine via phenylalanine hydroxylase in the cytosol of epidermal melanocytes provides the majority of the L-tyrosine pool for melanogenesis. In this context, it has been shown that the cofactor 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is produced de novo, recycled and regulated in both epidermal melanocytes and keratinocytes to control tyrosine hydroxylase, phenylalanine hydroxylase and tyrosinase activity. Inhibition of the enzymes by excessive 6BH4 levels is reversible with alpha-MSH by specific complex formation between 6BH4 and the hormone. This direct mechanism of alpha-MSH is supported by the presence of the entire POMC processing system in the melanosome indicating a receptor independent control of eumelanogenesis. Finally, the role of tyrosinase, TRP-1 and TRP2 is discussed in association with oxidative stress specifically related to hydrogen peroxide. These recent findings are based on detailed investigations of the depigmentation disorder vitiligo and Hermansky-Pudlák syndrome.     

Crystal structure and site-specific mutagenesis of pterin-bound human phenylalanine hydroxylase

The crystal structure of the dimeric catalytic domain (residues 118-424) of human PheOH (hPheOH), cocrystallized with the oxidized form of the cofactor (7,8-dihydro-L-biopterin, BH(2)), has been determined at 2.0 A resolution. The pterin binds in the second coordination sphere of the catalytic iron (the C4a atom is 6.1 A away), and interacts through several hydrogen bonds to two water molecules coordinated to the iron, as well as to the main chain carbonyl oxygens of Ala322, Gly247, and Leu249 and the main chain amide of Leu249. Some important conformational changes are seen in the active site upon pterin binding. The loop between residues 245 and 250 moves in the direction of the iron, and thus allows for several important hydrogen bonds to the pterin ring to be formed. The pterin cofactor is in an ideal orientation for dioxygen to bind in a bridging position between the iron and the pterin. The pterin ring forms an aromatic pi-stacking interaction with Phe254, and Tyr325 contributes to the positioning of the pterin ring and its dihydroxypropyl side chain by hydrophobic interactions. Of particular interest in the hPheOH x BH(2) binary complex structure is the finding that Glu286 hydrogen bonds to one of the water molecules coordinated to the iron as well as to a water molecule which hydrogen bonds to N3 of the pterin ring. Site-specific mutations of Glu286 (E286A and E286Q), Phe254 (F254A and F254L), and Tyr325 (Y325F) have confirmed the important contribution of Glu286 and Phe254 to the normal positioning of the pterin cofactor and catalytic activity of hPheOH. Tyr325 also contributes to the correct positioning of the pterin, but has no direct function in the catalytic reaction, in agreement with the results obtained with rat TyrOH [Daubner, S. C., and Fitzpatrick, P. F. (1998) Biochemistry 37, 16440-16444]. Superposition of the binary hPheOH.BH(2) complex onto the crystal structure of the ligand-free rat PheOH (which contains the regulatory and catalytic domains) [Kobe, B., Jennings, I. G., House, C. M., Michell, B. J., Goodwill, K. E., Santarsiero, B. D., Stevens, R. C., Cotton, R. G. H., and Kemp, B. E. (1999) Nat. Struct. Biol. 6, 442-448] reveals that the C2'-hydroxyl group of BH(2) is sufficiently close to form hydrogen bonds to Ser23 in the regulatory domain. Similar interactions are seen with the hPheOH.adrenaline complex and Ser23. These interactions suggest a structural explanation for the specific regulatory properties of the dihydroxypropyl side chain of BH(4) (negative effector) in the full-length enzyme in terms of phosphorylation of Ser16 and activation by L-Phe.     

Reduced 6,6,8-trimethylpterins. Preparation, properties and enzymic reactivities with dihydropteridine reductase, phenylalanine hydroxylase and tyrosine hydroxylase

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8-dihydro(6 H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8-trimethyl-5,6,7,8-tetrahydro(3 H)pterin was synthesised and the quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin derived from it is extremely stable with a half-life in 0.1 M Tris/HCl (pH 7.6, 25 degrees C) of 33 h. Quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin is not a substrate for dihydropteridine reductase but it is reduced non-enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 microM, pH 7.6, 25 degrees C when using quinonoid 6-methyl-7,8-dihydro(6 H)pterin as substrate. 6,6,8-Trimethyl-5,6,7,8-tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.     

Electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of -0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at -0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-dihydrobiopterin, sepiapterin, 2'-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82-1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and -0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3(*).     

High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin.

The crystal structures of the catalytic domain (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH) in its catalytically competent Fe(II) form and binary complex with the reduced pterin cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) have been determined to 1.7 and 1.5 A, respectively. When compared with the structures reported for various catalytically inactive Fe(III) forms, several important differences have been observed, notably at the active site. Thus, the non-liganded hPheOH-Fe(II) structure revealed well defined electron density for only one of the three water molecules reported to be coordinated to the iron in the high-spin Fe(III) form, as well as poor electron density for parts of the coordinating side-chain of Glu330. The reduced cofactor (BH4), which adopts the expected half-semi chair conformation, is bound in the second coordination sphere of the catalytic iron with a C4a-iron distance of 5.9 A. BH4 binds at the same site as L-erythro-7,8-dihydrobiopterin (BH2) in the binary hPheOH-Fe(III)-BH2 complex forming an aromatic pi-stacking interaction with Phe254 and a network of hydrogen bonds. However, compared to that structure the pterin ring is displaced about 0.5 A and rotated about 10 degrees, and the torsion angle between the hydroxyl groups of the cofactor in the dihydroxypropyl side-chain has changed by approximately 120 degrees enabling O2' to make a strong hydrogen bond (2.4 A) with the side-chain oxygen of Ser251. Carbon atoms in the dihydroxypropyl side-chain make several hydrophobic contacts with the protein. The iron is six-coordinated in the binary complex, but the overall coordination geometry is slightly different from that of the Fe(III) form. Most important was the finding that the binding of BH4 causes the Glu330 ligand to change its coordination to the iron when comparing with non-liganded hPheOH-Fe(III) and the binary hPheOH-Fe(III)-BH2 complex.     

Inactivation of tyrosine hydroxylase by reduced pterins

Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.     

Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS

5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.     

Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy.

Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe. The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes. Small shifts of the isotropic proton hyperfine coupling constants within the FAD's isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme. Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.     

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.     

Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase (eNOS), and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production (eNOS coupling). Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I (GTPCH). However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but whether DHFR is functionally important in maintaining eNOS coupling remains unclear. To investigate the mechanism by which DHFR might regulate eNOS coupling in vivo, we treated wild-type, BH4-deficient (hph-1), and GTPCH-overexpressing (GCH-Tg) mice with methotrexate (MTX), to inhibit BH4 recycling by DHFR. MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice. These effects were magnified in hph-1 but diminished in GCH-Tg mice. Attenuated eNOS activity was observed in MTX-treated hph-1 but not wild-type or GCH-Tg mouse lung, suggesting that inhibition of DHFR in BH4-deficient states leads to eNOS uncoupling. Taken together, these data reveal a key role for DHFR in regulating the BH4 vs BH2 ratio and eNOS coupling under conditions of low total biopterin availability in vivo.     

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.     

The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.     

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.     

Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin

Tryptophan hydroxylase oxidizes L-tryptophan to 5-hydroxy-L-tryptophan in the rate-determining step of serotonin biosynthesis. We have determined the X-ray crystal structure (1.7 A) of a truncated functional form of human tryptophan hydroxylase with the bound cofactor analogue 7,8-dihydro-L-biopterin, providing the first atomic-resolution information for the catalytic domain of this important enzyme. Comparison of the three-dimensional structures of all three members of the aromatic amino acid hydroxylase family--tyrosine hydroxylase, phenylalanine hydroxylase, and tryptophan hydroxylase--reveals important differences at the active sites.     

Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells

Tetrahydrobiopterin (BH4) is a member of the pterin family that has a core structure of pyrazino-2,3-d-pyrimidine rings. Because BH4 is an essential cofactor for the biosynthesis of nitric oxide (a major vasodilator), there is growing interest in BH4 biochemistry in endothelial cells (the cells that line blood vessels). BH4 is synthesized via de novo and salvage pathways from guanosine 5′-triphosphate (GTP) and 7,8-dihydrobiopterin, respectively, in animal cells. GTP cyclohydrolase-I (GTP-CH) is the first and rate-controlling enzyme in the de novo pathway. Available evidence shows that endothelial GTP-CH expression and BH4 synthesis are stimulated by a wide array of nutritional (phenylalanine and arginine), hormonal (insulin and estrogen), immunological (inflammatory cytokines including interleukin [IL]-1, interferon-γ, and tumor necrosis factor-α), therapeutic (statins and cyclosporin A), and endothelium-derived (basic fibroblast growth factor and H₂O₂) factors. In contrast, glucocorticoids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor [TGF]-β) inhibit endothelial BH4 synthesis. Because BH4 is oxidized to 7,8-dihydrobiopterin and 7,8-dihydropterin at physiological pH, endothelial BH4 homeostasis is regulated by both BH4 synthesis and its oxidation. Vitamin C, folate, and other antioxidants enhance endothelial BH4 bioavailability through chemical stabilization or scavenging of reactive oxygen species, thereby contributing to the maintenance of physiological homeostasis in the endothelium. New know ledge about the cellular and molecular mechanisms for the regulation of endothelial BH4 synthesis and bioavailability is beneficial for developing effective means to prevent and treat cardiovascular disorders, the leading cause, of death in developed nations.