Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases

Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.     

Comparison of Kinetic Properties Between Plant and Fungal Amine Oxidases

Abstract

Kinetic properties of novel amine oxidases isolated from a mold Aspergillus niger AKU 3302 were compared to those of typical plant amine oxidase from pea seedling (EC 1.4.3.6). Pea amine oxidase showed highest affinity with diamines, such as putrescine and cadaverine, while fungal enzymes oxidized preferably n-hexylamine and tyramine. All enzymes were inhibited by carbonyl reagents, copper chelating agents, some substrate analogs and alkaloids, but there were quite significant differences in the sensitivity and inhibition modes. Aminoguanidine, which strongly inhibited pea amine oxidases showed only little effect on fungal enzymes. Substrate analogs such as 1,5-diamino-3-pentanone and l-amino-3-phenyl-3-propanone, which were potent competitive inhibitors of pea amine oxidases, inhibited fungal enzymes much more weakly and non competitively. Also various alkaloids behaving as competitive inhibitors of pea amine oxidases inhibited the fungal enzymes non competitively. Very surprising was the potent inhibition of fungal enzymes by artificial substrates of pea amine oxidases, E- and Z-1,4-diamino-2-butene. The relationships between the different inhibition modes and possible binding at the active site are discussed.     

The depression of the synthesis of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors

The time courses of the synthesis of diamine oxidase in pea plants grown for 14 days either in the light or in the dark are similar with the highest increase in activity occurring in the cotyledons and in the shoots during the first 6 to 8 days. Plants grown in the dark showed a 2- to 3-fold higher enzyme activity than plants grown in the light. Pea diamine oxidase could bein vivo efficiently inhibited by substrate analogues 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone. The first compound inhibited proportionally to its concentration the growth of etiolated pea plants, but its instability makes an unequivocal interpretation of the results difficult. On the other hand, 1,5-diamino-3-pentanone a stable and more efficient diamine oxidase inhibitor depressed the growth of pea seedlings only at concentrations as high as 5 mM and 10 mM, at which the growth of cress seedlings not containing diamine oxidase was also strongly depressed. The results obtained indicate that tryptamine oxidation catalyzed by diamine oxidase is not involved in the main metabolic pathway leading from tryptophan to indoleacetate in pea plants.     

Probing the active site of pea seedlings amine oxidase with optical antipodes of sedamine alkaloids

Interactions of pea seedlings amine oxidase (PSAO, EC 1.4.3.6) with sedamine derivatives were studied. All compounds exhibited a competitive inhibition with the inhibition constants in the range 0.03-1.0 mM. The inhibition effect increased in the order allosedamine < sedamine < norallosedamine < norsedamine. The nor-derivatives are about five-fold stronger inhibitors and the allo-isomers are slightly weaker inhibitors than the others. Interestingly, the (-)-diastereomers of the studied sedamines were considerably stronger inhibitors than the (+)-antipodes. Absorption spectroscopy was used to differentiate between two known groups of competitive inhibitors of PSAO. A representative of substrate analogues, 1,5-diamino-3-pentanone, bleached the spectrum of the TPQ cofactor producing a very stable intermediate of the enzyme catalytic cycle that was only slowly converted to the product. On the other hand, the alkaloids did not perturb the spectrum of TPQ so they may interact with some other residue near the active site.     

1,4-Diamino-2-butyne as the mechanism-based pea diamine oxidase inhibitor.

1,4-Diamino-2-butyne is a mechanism-based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics Km = 1 mM like a substrate, but its interaction leads to time-dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4-diaminobutane and the competitive inhibitor 1,4-diamino-2-butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4-diamino-2-butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4-diamino-2-butyne is a mechanism-based inactivator with number of turnovers, r = 17 and characteristic constants K' = 0.32 mM and k(in) = 4.89 min-1.     

Peroxide induced activation of glycogen phosphorylase A activity in vascular smooth muscle

1-Phenoxy-2-propanone, 1-chloro-3-phenoxy-2-propanone, and 1-fluoro-3-phenoxy-2-propanone are competitive acetylcholinesterase inhibitors with K_I values of 30, 0.85, and 2.2 μM, respectively, compared to 2 mM for 4-phenyl-2-butanone. The substituent effect on inhibition suggests that these compounds bind by formation of a tetrahedral adduct and are transition state analogs.Other evidence supports this conclusion: N-benzyl-2-chloroacetamide and 1-phenoxy-2-propanol are poor inhibitors (K_I = 11 and >10 mM); 1-phenoxy-2-propanone and 1-chloro-3-phenoxy-2-propanone have K_I values 330 and 140 times smaller than K_m for corresponding substrates; and 1-chloro-3-phenoxy-2-propanone protects the enzyme against irreversible inhibition by CH₃SO₂F.     

Anti-coxsackievirus B3 activity of 2-amino-3-nitropyrazolo[1,5-a]pyrimidines and their analogs

A novel class of 2-amino-4-nitropyrazolo[1,5-a]pyrimidines has been identified as potent inhibitors of coxsackievirus B3 replication. The synthesis of these compounds is based on the regioselective reaction of 3,5-diamino-5-nitropyrazole with unsymmetrical beta-diketones at catalysis by hydrochloric acid leading to 2-amino-4-nitropyrazolo[1,5-a]pyrimidines as key steps.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

1,5-diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases.

1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.     

Identification of tertiary aminomethylenedioxy-propiophenones as urinary metabolites of safrole in the rat and guinea pig

Three nitrogen-containing metabolites of safrole (1-allyl-3,4-methylenedioxy-benzene) are excreted in the urine of rats and/or guinea pigs following oral or intraperitoneal administration. The major safrole basic ninhydrin-positive metabolites of the guinea pig and rat are 3-N-N-dimethylamino-1-(3′,4′-methylenedioxyphenyl)-1-propanone and 3-piperidyl-1-(3′,4′-methylenedioxyphenyl)-1-propanone, respectively. In addition, the rat also excretes the above N,N-dimethylaminoketone and trace amounts of 3-pyrrolidinyl-1-(3′,4′-methylenedioxyphenyl)-1-propanone. All three of these aminoketones decompose to form 1-(3′,4−methylenedioxyphenyl)-3-propen-1-one.     

Studies on Organic Insecticides

An improved synthesis of nereistoxin, 4-dimethylamino-1, 2-dithiolane (Ia), has been achieved in a 3-step sequence starting from 1, 3-bis (benzylthio)-2-propanone (IIa) or 2-phenyl-1, 3-dithian-5-one (IVa). By the similar route 4-amino-1, 2-dithiolane (Ib), 4-methylamino-1, 2-dithiolane (Ic) and two S-acyl derivatives (VIa, b) of dihydronereistoxin were synthesized and their insecticidal activities were tested.     

The structure and characterization of a modular endo-beta-1,4-mannanase from Cellulomonas fimi

The endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi is a modular plant cell wall degrading enzyme involved in the hydrolysis of the backbone of mannan, one of the most abundant polysaccharides of the hemicellulosic network in the plant cell wall. The crystal structure of a recombinant truncated endo-beta-1,4-mannanase from C. fimi (CfMan26A-50K) was determined by X-ray crystallography to 2.25 A resolution using the molecular replacement technique. The overall structure of the enzyme consists of a core (beta/alpha)8-barrel catalytic module characteristic of clan GH-A, connected via a linker to an immunoglobulin-like module of unknown function. A complex with the oligosaccharide mannotriose to 2.9 A resolution has also been obtained. Both the native structure and the complex show a cacodylate ion bound at the -1 subsite, while subsites -2, -3, and -4 are occupied by mannotriose in the complex. Enzyme kinetic analysis and the analysis of hydrolysis products from manno-oligosaccharides and mannopentitol suggest five important active-site cleft subsites. CfMan26A-50K has a high affinity -3 subsite with Phe325 as an aromatic platform, which explains the mannose releasing property of the enzyme. Structural differences with the homologous Cellvibrio japonicus beta-1,4-mannanase (CjMan26A) at the -2 and -3 subsites may explain the poor performance of CfMan26A mutants as "glycosynthases".     

Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.     

Phosphorylation of Raf-1 by Kinase Suppressor of Ras Is Inhibited by “MEK-Specific” Inhibitors PD 098059 and U0126 in Differentiating HL60 Cells

Determination of the involvement of MAP kinase cascades in signaling cell growth or differentiation is aided by the use of the inhibitors PD 098059 [2-(2′-amino-3′-methoxyphenyl)oxananphthalen-4-one] and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], believed to be MEK-specific kinase inhibitors. We report here that the activity of kinase suppressor of ras (KSR-1), a kinase upstream of raf-1, is inhibited by both these compounds at concentrations similar to those that inhibit MEK-1. Further, in HL60 cells induced to differentiate with 1,25-dihydroxyvitamin D₃ raf-1 and p90RSK, but not ERK1/2, are coregulated, and their expression as well as monocytic differentiation is inhibited in parallel by PD 098059. Thus, in this system raf-1 is phosphorylated by KSR-1, and PD 098059 as well as U0126 inhibits this phosphorylation. This suggests great caution in the interpretation of experiments that utilize these pharmacological inhibitors of kinase activity as evidence for a role for the MEK–ERK module in ras or raf-1 signaling.     

Novel ring transformation of 5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-isoxazole-4-carbaldehyde with 1,2-diaminobenzenes to 3-cyano-1,5-benzodiazepine C-nucleosides

Syntheses of 3-cyano-7- and 8-substituted-4-(beta-D-ribofuranosyl)-1H-1,5-benzodiazepines were reported. Treatment of isoxazole carbaldehyde with 1,2-diamino-4-nitrobenzene in chloroform gave a Schiffs base, 4-(2-amino-5-nitrophenyl)iminomethyl-5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)isoxazole in 82% yield with no trace of the other regioisomer. The cyclocondensation of the resulting Schiffs base in benzene containing trifluoroacetic acid (TFA) gave 3-cyano-8-nitro-4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1H-1,5-benzodiazepine in 49% yield. The same reaction of isoxazole carbaldeyde with 1,2-diamino-4-methoxy- and 4-chlorobenzenes afforded the corresponding Schiffs bases. Extending the reaction time for Schiffs base gave the corresponding cyanobenzodiazepines in good yields. Debenzoylation of the compounds with sodium methoxide produced deprotected C-nucleosides.     

Interaction of L- and D-3-amino-1-chloro-2-pentanone with gamma-glutamylcysteine synthetase

The optical isomers of 3-amino-1-chloro-2-pentanone, which are the alpha-chloroketone analogs of L- and D-alpha-aminobutyrate, were synthesized and found to be highly potent irreversible inactivators of gamma-glutamylcysteine synthetase. These chloroketones are 20 to 30 times more active than L-2-amino-4-oxo-5-chlorpentanoate. L- and D-Glutamate, in the presence of Mg2+ or Mn2+, protect the enzyme against inactivation. The enzyme is almost completely inhibited by cystamine under conditions in which 0.5 mol of this compound is bound/mol of enzyme. Treatment of the enzyme with cystamne, which produces inhibition that is reversible by dithiothreitol, prevents the interaction of the new chloroketones, L-2-amino-4-oxo-5-chloropentanoate and methionine sulfoximine with the enzyme. The findings suggest that a sulfhydryl group at the active site interacts with the chloroketones and with cystamine and that the chloroketone inhibitors and cystamine bind to the enzyme as glutamine analogs. The data also suggest that a gamma-glutamyl-S-enzyme intermediate may be formed in the reaction catalyzed by this enzyme.     

Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, beta-1,4-galactosylated and alpha-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (-2-1-+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites -4 and -3 of Chit42, and at the subsite -4 of Chit33. Fucose units can be accepted as a branch at the putative -3 and -4 sites of Chit33 and as a branch point at -3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.     

Novel alpha-L-fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae).

The extract of bark of Angylocalyx pynaertii (Leguminosae) was found to potently inhibit mammalian alpha-L-fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 1-deoxymannojirimycin (DMJ) and 2,5-imino-1,2,5-trideoxy-D-mannitol (6-deoxy-DMDP). Among them, eight sugar-mimic alkaloids showed the potent inhibitory activity towards bovine epididymis alpha-L-fucosidase and their Ki values are as follows: 6-deoxy-DMDP (83 microM), 2,5-imino-1,2,5-trideoxy-L-glucitol (0.49 microM), 2,5-dideoxy-2,5-imino-D-fucitol (17 microM), 2,5-imino-1,2,5-trideoxy-D-altritol (3.7 microM), DMJ (4.7 microM), N-methyl-DMJ (30 microM), 6-O-alpha-L-rhamnopyranosyl-DMJ (Rha-DMJ, 0.06 microM), and beta-L-homofuconojirimycin (beta-HFJ, 0.0053 microM). We definitively deduced the structural requirements of inhibitors of alpha-L-fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of alpha-L-fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of L-fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of L-fucose generates extremely powerful inhibition of alpha-L-fucosidase. The replacement of the methyl group of beta-HFJ by a hydroxymethyl group reduced its inhibitory potential about 80-fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of L-fucose does not appear to be important for the inhibition. Interestingly, Rha-DMJ was a 70-fold more potent inhibitor of alpha-L-fucosidase than DMJ. This implies that the lysosomal alpha-L-fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates.     

Asymmetric catalysis 87. Enantioselective allylation of 1,5-dimethylbarbituric acid with Pd catalysts and new optically active PN ligands

The new PN ligands 5, 6 and 7 were prepared by Schiff base condensation of 2-formylphenyl(diphenyl)phosphine (1) with the optically active amines (R)-(−)-2-aminobutanol (2), (S)-(+)-2-aminobutanol (3) and (1S,2S)-2-amino- 1-phenyl-1,3-propanediol (4). These new ligands were used in the Pd catalysed allylation of 1,5-dimethylbarbituric acid with allylacetate. 5-Allyl-1,5-dimethylbarbituric acid was obtained with an optical induction of up to 12.7% ee.