Peroxidase catalysed aerobic degradation of 5,6,7,8-tetrahydrobiopterin at physiological pH

The aerobic degradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(6H)pterin, and this finally rearranges to 7,8-dihydroxanthopterin. Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3H)pterin. The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 microM and kcat of 22.5 s-1. The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer. By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)-7,8-dihydro(6H)biopterin Km = 1.3 microM and kcat = 22.8 s-1; with (6S)-7,8-dihydro(6H)biopterin Km = 13.5 microM and kcat = 51.6 s-1; and with (6RS)-7,8-dihydro(6H)neopterin Km = 19.2 microM and kcat = 116 s-1.     

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.     

Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one with dihydropteridine reductase.

The Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one were determined for dihydropteridine reductase (EC 1.6.99.10) from two sources. The parameters of the pterin are of the same order as those of the most effective substrates of dihydropteridine reductase. The Km values of the pterin are one order of magnitude smaller than those of the pyrimidinone, although the kcat. values are of the same order.     

Tetrahydrobiopterin analogues: solution conformations of 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyltetrahydropterins as determined by proton nuclear magnetic resonance

The conformation of tetrahydrobiopterin analogues in aqueous solution at 23 degrees C has been determined by analyzing the 200-MHz 1H NMR spectral parameters of the enzymatically active 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyl-5,6,7,8-tetrahydropterins. Each of these cofactors, with the exception of the cis-6,7-dimethyl isomer, exhibited an unusually small trans 6H-7H spin-spin coupling (8.5-9.1 Hz). An empirical equation that accounts for the effects of substituent electronegativity and orientation on vicinal couplings [Haasnoot, C. A. G., deLeeuw, F. A. A. M., & Altona, C. (1980) Tetrahedron 36, 2783-2792] predicted this coupling to be 11.3-11.6 Hz. We attribute the discrepancy between the calculated and experimentally observed values of this coupling to hyperconjugation of the axially oriented C7-H bond with the pi orbital of the vinylogous amide protein of the pterin ring (N8-C8a = C4a-C4 = O) rather than conformational averaging. The trans 6H-7H interproton distance in the 6-methyl analogue is calculated to be 3.0 A from the measured decrease in the spin-lattice relaxation rate of the axially oriented C7 proton after specific deuteration at C6. This is consistent with the single-conformer interpretation. Chemical shift comparisons of the methyl resonances of these analogues, NOE measurements from selectively deuterated analogues, and the differential sensitivities of axially vs. equatorially disposed ring protons to protonation at N5 all indicate that (i) the methyl substituents at both the C6 and C7 positions markedly prefer equatorial-like orientations and (ii) the tetrahydropterin ring is, with the exception of a pronounced pucker at C6, nearly planar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of dihydropteridine reductase from Pseudomonas species.

Dihydropteridine reductase isolated from the bacterium Pseudomonas species (ATCC 11299a) has been purified approximately 450-fold byammonium sulfate precipitation and diethylaminoethyl-cellulose chromatographic procedures. The preparation is at least 80% pure as judged by polyacrylamide gels. Its molecular weight was determined to be about 44,000. Both dihydropteridine reductase and phenylalanine hydroxylase activities were found to be higher in cells adapted to a medium containing L-phenylalanine or L-tyrosine as the sole carbon source than in those grown in L-asparagine. The substrate of the reductase is quinonoid dihydropteridine, and the product is tentatively identified as a tetrahydropteridine through its ability to serve as a cofactor for phenylalanine hydroxylase. The enzyme shows no marked specificity for the pteridine cofactor that occurs naturally in this organism, L-threo-neopterin. The pH optimum for the reductase is 7.2, and nicotinamide adenine dinucleotide, reduced form, is the preferred cosubstrate. Inhibition of the reduced and untreated enzyme by several sulfhydryl reagents was observed. A metal requirement for the reductase could not be demonstrated. Dihydropteridine reductase was found to be inhibited by aminopterin in a competitive manner with respect to the quinonoid dihydro form of 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine.     

Inactivation of dihydropteridine reductase (human brain) by platinum(II) complexes

Potassium tetrachloroplatinate (K2PtCl4) inactivates dihydropteridine reductase from human brain in a time-dependent and irreversible manner. The inactivation has been followed by measuring enzyme activity and fluorescence changes. The enzyme is completely protected from inactivation by NADH, the pterin cofactor [quinonoid 6-methyl-7,8-dihydro(6H)pterin] and dithiothreitol. Evidence is presented that K2PtCl4 reacts at the active site and that (a) thiol group(s) is involved in, or is masked by, this reaction. K2PtCl4 is a stronger inhibitor of human brain dihydropteridine reductase that cis- and trans-diaminodichloroplatinum, cis-dichloro[ethylenediamine]platinum and K4Fe(CN)6, whereas H2PtCl6 is considerably weaker and (Ph3P)3RhCl is inactive.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Studies on the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase

The uncoupled portion of the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase can be described by the same model as we have recently derived for the fully uncoupled reaction (Davis, M.D. and Kaufman, S. (1989) J. Biol. Chem.264, 8585–8596). Although essentially no hydrogen peroxide is formed during the fully coupled oxidation of tetrahydrobiopterin or 6-methyltetrahydropterin by phenylalanine hydroxylase when phenylalanine is the amino acid substrate, significant amounts of hydrogen peroxide are formed during the partially uncoupled oxidation of 6-methyltetrahydropterin whenpara-fluorophenylalanine orpara-chlorophenylalanine are used in place of phenylalanine. Similarly, during the partially uncoupled oxidation of the unsubstituted pterin, tetrahydropterin, even in the presence of phenylalanine, hydrogen peroxide formation is detected. The 4a-carbinolamine tetrahydropterin intermediate has been observed during the fully uncoupled tyrosine-dependent oxidations of tetrahydropterin and 6-methyltetrahydropterin by lysolecithin-activated phenylalanine hydroxylase, suggesting that this species is also a common intermediate for uncoupled oxidations by this enzyme.Abbreviations BH₄ 6-[dihydroxypropyl-(L-erythro)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) - 6MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PH₄ 5,6,7,8-tetrahydropterin - BH₃OH 4a-hydroxytetrahydropterin (4a-carbinolamine) - qBH₂ quinonoid dihydrobiopterin - q6MPH₂ quinonoid dihydro-6-methylpterin - qPH₂ quinoid dihydropterin - PAH phenylalanine hydroxylase - DHPR dihydropteridine reductase - PHS phenylalanine hydroxylase stimulating enzyme which is 4a-carbinolamine dehydratase - SOD superoxide dismutase - HPLC high performance liquid chromatography - R.T. retention time Special issue dedicated to Dr. Santiago Grisolia.     

BIOPTERIN V. DE NOVO SYNTHESIS OF DIHYDROBIOPTERIN: EVIDENCE FOR ITS QUINONOID STRUCTURE AND LACK OF DEPENDENCE OF ITS REDUCTION TO TETRAHYDROBIOPTERIN ON DIHYDROFOLATE REDUCTASE

Samples of quinonoid-l -erythrodihydrobiopterin (q-BH₂) and quinonoid-6-methyl-dihydro-pterin (q-6-MPH₂) were prepared by oxidation of l -erythro-5,6,7,8-tetrahydrobiopterin (BH₄) and 5,6,7,8-tetrahydro-6-methylpterin (6-MPH₄) and separated from ^D-erythro-7,8-dihydrobiopterin (7,8-BH₂) and 6-methyl-7,8-dihydropterin (7,8-6-MPH₂) as well as from the tetrahydropterins on phosphocellulose column by high-pressure liquid chromatography. The quinonoid dihydropterins were identified and quantitated by scan of their ultraviolet absorption and fluorescence emission spectra through their rearrangement to their 7,8-tautomer and also by gas chromatography of their rapidly synthesized trimethylsilyl derivative. Identification was also achieved by the enzymatic reduction of [³H]q-BH²to [³H]BH₄ by dihydrofolate reductase (DHFR). Direct proof for the enzymatic synthesis of the q-BH² from GTP or from 2-amino-6-(5′-triphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimi-dine (FPyd-P₃) was obtained by isolation of the compound which was identical in all respects to the q-BH₂ obtained by chemical synthesis from BH₄. The reduction of enzymatically synthesized q-BH² by dihydropteridine reductase (DHPR) to BH⁴ was not inhibited by methotrexate (MTX). When the enzymatically synthesized q-BH² was converted to 7,8-BH₂, it was reduced only by DHFR. This reduction, however, was inhibited by MTX. On the biosynthetic pathway from GTP to dihydrobiopterin, the enzyme responsible for the appearance of the quinonoid structure is the d -erythro-dihydroneopterin triphosphate synthetase, the product of which (quinonoid d -erythro-dihydroneopterin triphosphate) is converted to quinonoid dihydrobiopterin by l -erythro-dihydrobiopterin synthetase. Experiments in vivo established that DHFR does not participate in the reduction of dihydrobiopterin to tetra-hydrobiopterin when the former is synthesized from GTP de novo. MTX at 5 × 10^?6M exerted no inhibition on the reduction of the biosynthetic dihydrobiopterin to tetrahydrobiopterin in vivo, yet completely inhibited the reduction of intraventricularly injected tritiated dihydrofolate ([³H]FH₂) to tritiated tetrahydrofolate ([³H]FH₄).     

The oxidation of apomorphine and other catechol compounds by horseradish peroxidase: relevance to the measurement of dihydropteridine reductase activity

It has been reported by Shen et al. (Shen, R.-S., Smith, R.V., Davis, P.J. and Abell, C.W. (1984) J. Biol. Chem. 259, 8894-9000) that apomorphine and dopamine are potent, non-competitive inhibitors of quinonoid dihydropteridine reductase. In this paper we show that apomorphine, dopamine and other catechol-containing compounds are oxidized rapidly to quinones by the horseradish peroxidase-H2O2 system which is used to generate the quinonoid dihydropterin substrate. These quinones react non-enzymatically with reduced pyridine nucleotides, depleting the other substrate of dihydropteridine reductase. When true initial rates of dihydropteridine reductase-dependent reduction of quinonoid dihydropterins are measured, neither apomorphine nor any other catechol-containing compound that has been tested has been found to inhibit dihydropteridine reductase.     

猪肝二氢蝶啶还原酶的提取及动力学研究

本文报导了从猪肝中提取二氢蝶啶还原酶[Ecl.6.99.7]的方法,提取百分率达30％左右。以DMPH_4为底物,分别以NADH和NADPH为辅酶,测定了该酶的动力学,发现它对NADH具有一定的特异性[Km(NADH)Vmax(NADPH)]。不同的金属离子对该酶活性影响的程度有很大的差异。     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Pyrimidodiazepine, a ring-strained cofactor for phenylalanine hydroxylase

Homologues of 6-methyl-7,8-dihydropterin (6-Me-7,8-PH2) and 6-methyl-5,6,7,8-tetrahydropterin (6-Me-PH4), expanded in the pyrazine ring, were synthesized to determine the effect of increased strain on the chemical and enzymatic properties of the pyrimidodiazepine series. 2-Amino-4-keto-6-methyl-7,8-dihydro-3H,9H-pyrimido[4,5-b] [1,4]diazepine (6-Me-7,8-PDH2) was found to be more unstable in neutral solution than 6-Me-7,8-PH2. Its decomposition appears to proceed by hydrolytic ring opening of the 5,6-imine bond, followed by autooxidation. 6-Me-7,8-PDH2 can be reduced, either chemically or by dihydrofolate reductase (Km = 0.16 mM), to the 5,6,7,8-tetrahydro form (6-Me-PDH4). This can be oxidized with halogen to quinoid dihydropyrimidodiazepine (quinoid 6-Me-PDH2), which is a substrate for dihydropteridine reductase (Km = 33 microM). Whereas quinoid 6-methyldihydropterin was found to tautomerize to 6-Me-7,8-PH2 in 95% yield in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, quinoid 6-Me-PDH2 gives only 53% 6-Me-7,8-PDH2, the remainder decomposing via an initial opening of the diazepine ring. Additional evidence for the extra strain in the pyrimidodiazepine system is the cyclization of quinoid 6-N-(2'-aminopropyl)divicine to quinoid 6-Me-PH2 in 57% yield in 0.1 M Tris-HCl, pH 7.4. By comparison, no quinoid 6-Me-PDH2 is formed from the homologue quinoid 6-N-(3'-aminobutyl)divicine. A small (2%) yield of 6-Me-PDH4 is found if the unstable C4a-carbinolamine intermediate is trapped by enzymatic dehydration and reduction. Although phenylalanine hydroxylase utilizes 6-Me-PDH4 (Km = 0.15 mM), the maximum velocity of tyrosine production is 20 times slower than that with 6-Me-PH4, indicating that a ring opening reaction is not a rate-limiting step in the hydroxylase pathway. Further, the maximum velocities of 2,5,6-triamino-4(3H)-pyrimidinone, 2,6-diamino-5-(methylamino)-4(3H)-pyrimidinone, and 2,6-diamino-5-(benzylamino)-4(3H)-pyrimidinone span a 35-fold range. These cofactors would theoretically form the same oxide of quinoid divicine if oxygen activation involves a carbonyl oxide intermediate. Thus, the limiting step is also not transfer of oxygen from this hypothetical intermediate to the phenylalanine substrate.     

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.     

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.     

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.     

Synthesis, antioxidant and toxicological study of novel pyrimido quinoline derivatives from 4-hydroxy-3-acyl quinolin-2-one

A series of novel pyrimido and other fused quinoline derivatives like 4-methyl pyrimido [5,4-c]quinoline-2,5(1H,6H)-dione (4a), 4-methyl-2-thioxo-1,2-dihydropyrimido [5,4-c]quinoline-5(6H)-one (4b), 2-amino-4-methyl-1,2-dihydropyrimido [5,4-c]quinolin-5(6H)-one (4c), 3-methylisoxazolo [4,5-c]quinolin-4(5H)-one (4d), 3-methyl-1H-pyrazolo [4,3-c]quinoline-4(5H)-one (5e), 5-methyl-1H-[1,2,4] triazepino [6,5-c]quinoline-2,6(3H,7H)-dione (5f), 5-methyl-2-thioxo-2,3-dihydro-1H-[1,2,4]triazepino [6,5-c]quinolin-6(7H)-one (5 g) were synthesized regioselectively from 4-hydroxy-3-acyl quinolin-2-one 3. They were screened for their in vitro antioxidant activities against radical scavenging capacity using DPPH(), Trolox equivalent antioxidant capacity (TEAC), total antioxidant activity by FRAP, superoxide radical (O(2)(°-)) scavenging activity, metal chelating activity and nitric oxide scavenging activity. Among the compounds screened, 4c and 5 g exhibited significant antioxidant activities.     

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.     

Synthesis of menaquinone-2 derivatives as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of a number of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) derivatives. The 2-des-methyl and 2-ethyl-MK-2 derivatives had very low activity as substrates. The 6- or 7-methyl-MK-2 derivatives and (6,7)-chloro-MK-2 were relatively high Vmax substrates with Km values increased over that seen for K-2. The 5- or 8-methyl-MK-2 derivatives were low Vmax substrates but also demonstrated low Km values. Although these observations suggested that 5-methyl-MK-2 might be a competitive inhibitor of the carboxylation reaction, it was not an effective inhibitor of either phylloquinone or 6-methyl-MK-2-dependent carboxylation.