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PubMed2003

Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.

Animals↗
PubMed1997

The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.

Kinetic parameters and primary deuterium isotope effects have been determined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala133Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the natural substrate, quinonoid (6R)-l-erythro-dihydrobiopterin (qBH2) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7-diMePtH2). Mutation at either Tyr146 or Lys150 resulted in pronounced changes in kinetic parameters and isotope effects for both pterin substrates, confirming a critical role for these residues in enzyme-mediated hydride transfer. By contrast, the Ala133Ser mutant was practically indistinguishable from wild-type enzyme. The changes observed, however, were quite different for the two pterin substrates. Thus, kcat for q-6,7-diMePtH2 decreased across the series of mutants from a value of 150 s-1 for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely, kcat for qBH2 increased 3-11-fold across the same series of mutants from the wild-type value of 23 s-1. For both pterin substrates, the Km (KPt) increased several orders of magnitude upon mutation of Tyr146 or Lys150, with the greater relative increase using qBH2. Significant primary deuterium isotope effects on kcat (Dkcat) and kcat/KPt (D(kcat/KPt)) observed for the Tyr146 and Lys150 mutants varied depending on the pterin substrate used and ranged up to a maximum value of 5.5-6. For qBH2, where Dkcat < Dkcat/KPt was consistently observed, the rate determining step is ascribed to release of the tetrahydropterin product. For q-6,7-diMePtH2, where in all cases Dkcat = Dkcat/KPt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer. Modeling studies in which qBH2 was docked into the binary E:NADH complex provide a structural rationale for the observed differences between the two pterin substrates. The natural substrate, qBH2, displays a higher affinity for the enzyme active site, presumably due to interaction of the dihydroxypropyl side chain of the substrate with a polar loop of residues containing Asn186, Ser189, and Met190. The location of this loop within the three-dimensional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) family, which includes dihydropteridine reductase.

Animals↗
PubMed1990

Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.

Chromatography, High Pressure Liquid↗
PubMed2002

Pteridine derivatives as modulators of oxidative stress.

Pteridine derivatives which have a widespread occurrence in nature have been investigated upon their interactions with free radicals and free radical mediated reactions utilizing a number of different experimental systems. Searching for biological functions, which are still unknown for the majority of pteridine compounds, the effect of pteridines in systems like luminol-induced chemiluminescence, enzyme activity, DNA photodamage, EPR experiments or radical induced injury--just to name a few--have been investigated. The general view during the initial phase of investigations on this special field was, that reduced pterins, i. e., tetra- as well as dihydropterins, generally act as radical scavengers, while aromatic pterins, if not inactive, exert radical promoting activity. Meanwhile the data available provide a more complex view: pteridines of all oxidation states have been shown to act anti- or prooxidatively, depending on the special conditions of the experiment. The reason is that reduced pterins, besides of being scavengers of free radicals, also are strongly reducing agents and therefore, in the presence of transition metal ions promote Fenton chemistry. Aromatic pterins have been described as inhibitors or substrates of enzymes involved--in vitro and in vivo--in free radical generation. Together with the unknown local concentrations of, e.g., neopterin and dihydroneopterin occurring in vivo, these reasons make it impossible to unequivocally predict a physiological net effect of pterins of different oxidation states concerning free radical mediated reactions in real biological systems.

Animals↗
PubMed1984

Folic acid does not inactivate xanthine oxidase.

In response to a recent report (Lewis, A.S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15) that folic acid was a potent inactivator of xanthine oxidase, the details of this apparent inactivation were studied. In confirmation, we also found that commercially available folic acid produced a time-dependent progressive inhibition (apparent inactivation) of xanthine oxidase. A plot of the pseudo-first order rate constant of the decay of enzyme activity versus the concentration of folic acid resulted in a straight line. This indicated that the progressive inhibition was caused by a slow second order combination of an inhibitor with the enzyme. The second order rate constant for this association (slope of replot) was 5.7 X 10(3) M-1 S-1. The slowness of this constant together with the observation that complete inactivation did not occur suggested that the progressive inhibition might be due to the slow binding of a high affinity contaminant. This was corroborated by the finding that the association constant was decreased to 1.6 X 10(2) M-1 S-1 after partially purifying the folic acid. The compound most likely to be producing this inhibition is pterin aldehyde (2-NH2-4-OH-pteridine-6-aldehyde), a photolytic breakdown product of folic acid. Pterin aldehyde was found to be a progressive inhibitor of xanthine oxidase with an association constant of 2.2 X 10(5) M-1 S-1. When the apparent association constants of commercial and purified folic acid were adjusted to reflect the pterin aldehyde content (3.6% and 0.2%, respectively), they became similar to the association constant of pterin aldehyde. Thus, it seems that the apparent inactivation of xanthine oxidase by folic acid was caused by the slow binding of contaminating pterin aldehyde.

Animals↗
PubMed1994

Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity.

Activation by phenylalanine and reduction by the co-factor (6R)-tetrahydrobiopterin (BH4) are required for formation of active liver phenylalanine hydroxylase. This work describes effects of the activation and redox state on substrate and effector recognition of this enzyme, it establishes relationships among the pterin and phenylalanine binding sites on the different forms of the enzyme, and it provides a quantitative description of the enzyme's presumptive regulatory and catalytic sites. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-deaza-6-methyltetrahydropterin were found to bind to unactivated phenylalanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site on the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. In the transition from unactivated to activated enzyme, phenylalanine and pterin binding is modified, a new site for phenylalanine is formed, and the pterin site is replaced by a site of greatly decreased affinity for BH4 and BH2, one which does not appear to recognize the dihydroxypropyl side chain of BH4 and BH2. The pterin- and phenylalanine-binding sites on activated phenylalanine hydroxylase appear to be part of the enzyme's active site. Despite large effects on substrate binding, neither chelator binding ability nor solvent accessibility of the iron are affected by activation; activation appears to affect the nearby environment of the enzyme's iron but not the iron itself. Studies of oxidized and reduced phenylalanine hydroxylase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.

Animals↗
PubMed1994

Chiral lumazines: preparation, properties, enantiomeric separation.

Optically active lumazines (biolumazine, dictyolumazine, monalumazine, and neolumazine) are prepared from the corresponding pterins by enzymatic reaction, using pterin deaminase excreted by Dictyostelium discoideum. The fluorescence properties, circular dichroism spectra, and chromatographic behavior of these lumazines are studied. D- and L-enantiomers of biolumazine, dictyolumazine, and monalumazine are separated using a chiral flavoprotein column. This column also separates the enantiomeric pterins of the threo form: monapterin and dictyopterin. However, the column does not separate the enantiomeric pterins of the erythro form: neopterin and biopterin. By coupling a reverse-phase column to the flavoprotein column, the separation of pterins and lumazines in function of their hydrophobicity, as well as the separation of the diastereomers, is achieved. This coupled achiral/chiral high-performance liquid chromatography method enables determination of the stereoconfiguration of natural lumazines by comparison with optically pure compounds. A lumazine derivative, present in the extracellular medium of Dictyostelium discoideum, is identified as D-dictyolumazine, i.e., 6-(D-threo-1,2-dihydroxypropyl)-lumazine.

Animals↗
PubMed2002

Quantitative analysis of the behavior of Dictyostelium discoideum amoebae: stringency of pteridine reception.

A convenient, sensitive, quantitative assay for the measurement of chemotaxis of populations of D. discoideum vegetative amoebae is presented. A strategy for determining the boundary of the bulk of a population of migrating amoebae was devised and is described. This assay employs a dynamic gradient and is independent of deaminase activity. Measurements of chemoattractant capabilities of various pteridines, folates, and mixtures of folate fragments are reported. 2-Amino 4-quinazolinone, a pterin analog without the pyrazine ring nitrogens, is chemotactic. Lumazine, deaminated pterin, inhibits chemotaxis towards pterin but not towards folic acid. Deaminofolic acid is a chemoattractant as are mixtures of lumazine plus aminobenzoylglutamic acid or deaminopteroic acid plus various amino acids. Separately, the components of these mixtures exhibit no ability to stimulate chemotaxis. These mixtures are of fragments that together comprise most of the folate structure. Our results are in accord with separate receptors for pterin vs. folic acid and with a high stringency for pterin reception but a relative tolerance for folate reception. The possibility of using such mixtures to investigate the requirements of various parts of the folate structure for competent signalling is discussed.

Animals↗
PubMed1992

Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.

The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗
PubMed2003

Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys.

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both L-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the Fe(II) site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).

Absorptiometry, Photon↗
PubMed2004

Enhancement of folates in plants through metabolic engineering.

Humans depend on plants as a major source of dietary folates. Inadequate dietary levels of the vitamin folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer. The biofortification of folate levels in food crops is a target for metabolic engineering. Folates are synthesized de novo from pterins and para-amino benzoic acid, which are subsequently combined to form dihydropteroate, the direct precursor to dihydrofolate. We postulated that GTP cyclohydrolase-1, which catalyzes the first committed step in pterin biosynthesis, was a rate-limiting step in pterin synthesis in plants and, therefore, in folate synthesis. On this basis, we proposed that the expression of an unregulated bacterial GTP cyclohydrolase-1 in plants would increase pterin biosynthesis with a concomitant enhancement of folate levels. The folE gene encoding GTP cyclohydrolase-1 was cloned from Escherichia coli and introduced into Arabidopsis thaliana through plant transformation. The expression of bacterial GTP cyclohydrolase-1 in transgenic Arabidopsis resulted in a 1,250-fold and 2- to 4-fold enhancement of pterins and folates, respectively. These results helped to identify other potential factors regulating folate synthesis, suggesting ways to further enhance folate levels in food crops.

Arabidopsis↗
PubMed1999

Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase.

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.

Arginine↗
PubMed1991

Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities.

The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.

Animals↗
PubMed1990

DNA photorepair: chromophore composition and function in two classes of DNA photolyases.

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.

Biopolymers↗
PubMed1994

Regulation of rat liver phenylalanine hydroxylase. III. Control of catalysis by (6R)-tetrahydrobiopterin and phenylalanine.

Effects of phenylalanine and di- and tetrahydropterins on presteady-state and steady-state catalytic behavior of rat liver phenylalanine hydroxylase are analyzed. From this and previous work (Shiman, R, Xia, T., Hill, M., and Gray, D.(1994) J. Biol. Chem. 269, 24647-24656), which analyzed binding of the same compounds to the enzyme in the absence of catalysis, a model of phenylalanine hydroxylase regulation is proposed. The mechanism appears novel in that 1) one substrate, phenylalanine, is a positive effector (activator), 2) a second substrate, (6R)-tetrahydrobiopterin (BH4), is a negative effector that blocks phenylalanine activation by forming an inactive BH4.enzyme complex, and 3) the BH4.enzyme complex sequesters BH4 and controls its metabolic availability. Reaction progress curves showing regulatory effects of BH4, 7,8-dihydrobiopterin (BH2), and phenylalanine are fit by the model with high precision. Data are presented that the high affinity pterin-binding site on unactivated phenylalanine hydroxylase is the pterin site that regulates catalysis. Occupancy of this site by BH4 or BH2 causes non-cooperative, linear inhibition of phenylalanine activation of the enzyme. All inhibitory effects of BH4 appear due to its binding at the pterin regulatory site on unactivated enzyme. BH2 inhibits by binding at the active site as well as the pterin regulatory site. 6-Methyltetrahydropterin also appears to bind at the pterin regulatory site, but its effect is only seen at high phenylalanine concentrations. Using kinetic constants measured in this and earlier work, quantitative effects of phenylalanine and BH4 regulation on the rate of the phenylalanine hydroxylase reaction in vitro and in vivo are calculated. The effects of formation of the BH4.enzyme complex on free BH4 concentration, on enzyme activity, and on regulation of the rate of phenylalanine hydroxylation in liver are discussed.

Animals↗
PubMed1998

Recognition and interaction of small rings with the ricin A-chain binding site.

Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings.

Adenine↗
PubMed1983

Influence of some pteridines on pineal 5-methoxyindole synthesis in male Wistar rats periodically exposed to either white or red light.

In previous investigations the change of circadian rhythmicity in pineal melatonin/5-methoxytryptophol synthesis of rats periodically exposed to red light was similar to that in pineals of rats incubated with pterin-6-aldehyde. These experiments were, however, performed with rats of different age and in different periods of the year. In the present study these two factors influencing pineal indole metabolism have been combined the experiments being carried out in rats aged 28 days and during the same day in the month of January. It was observed that under influence of red light the peak of melatonin/5-methoxytryptophol synthesis shifted towards daytime, whereas incubation with pterin-6-aldehyde did not cause such a shift. If under different experimental conditions the mean amount of melatonin/5-methoxytryptophol which was formed over a 24 hour period was compared, it appeared that pineals of rats exposed to white light incubated with reduced neopterin but not pineals incubated with pterin-6-aldehyde behave in this respect similar to pineals of rats exposed to red light. However, if the ratio between melatonin/5-methoxytryptophol and 5-methoxytryptamine is calculated pineals of white light exposed rats incubated in pterin-6-aldehyde behaved very similar to the pineals of rats exposed to red light. Although the role of pteridines remains obscure, it appears that the parameters 2. circadian rhythmicity and 2. the amount of 5-methoxyindoles and 3. the ratio between these indole derivatives might be of importance in analyzing their physiological effects. The influence of application of light of different wavelengths and year rhythmicity is discussed.

Animals↗
PubMed1990

Isolation and characterization of a second molybdopterin dinucleotide: molybdopterin cytosine dinucleotide.

The pterin cofactor (bactopterin) in the molybdoenzyme CO dehydrogenase isolated from Pseudomonas carboxydoflava has previously been shown to differ from molybdopterin in molecular mass, phosphate content, stability, and other properties, implying a novel structure. The structure of the CO dehydrogenase pterin has been investigated in the present studies by alkylation and isolation of the carboxamidomethyl derivative. The alkylated pterin was identified as [di-(carboxamidomethyl)]molybdopterin cytosine dinucleotide on the basis of its absorption properties and by degradation with nucleotide pyrophosphatase yielding carboxamidomethylmolybdopterin and CMP. Further treatment of these products with alkaline phosphatase produced species with absorption and chromatographic properties identical to those of the corresponding dephospho compounds. Molybdopterin cytosine dinucleotide is the second molybdopterin variant to be structurally characterized. The fact that molybdopterin cytosine dinucleotide and molybdopterin guanine dinucleotide contain molybdopterin in their structure shows that the pterin moiety, with its unique dithiolene-containing sidechain, is a structural element which is common to the organic portion of the molybdenum cofactors of many molybdoenzymes.

Aldehyde Oxidoreductases↗