An active-site titrant for human tissue-type plasminogen activator.

The reaction of recombinant tissue-type plasminogen activator with the inverse substrate 4-amidino-2-nitrophenyl 4'-anisate results in the rapid release of the chromogen 4-amidino-2-nitrophenol and the accumulation of the relatively stable 4-anisoyl-enzyme. Spectrophotometric monitoring of the reaction enables the operational molarity of the enzyme to be determined.     

Differentiation of feruloyl esterases on synthetic substrates in alpha-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays

4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubová, J. Szemesová, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

Metabolism of phenol,chloro- and nitrophenols by the Penicillium strain Bi 7/2 isolated from a contaminated soil

The Penicillium strain Bi 7/2 able to grow on phenol as sole source of carbon and energy was isolated from a contaminated soil in Bitterfeld (East Germany). The strain is adapted to high phenol concentrations. Spores germinated still at a phenol concentration of 1.5 g/l. Phenol is degraded by the ortho-pathway with catechol as first intermediary product. The Penicillium strain metabolizes 4-, 3- and 2-chlorophenol with decreasing rates with phenol or glucose as cosubstrate. In the case of 4-chlorophenol 4-chlorocatechol was detected as intermediary product, further degraded as indicated by release of about 35% of the bound chlorine of the aromatic molecule. The strain also cometabolically metabolizes 4-, 3- and 2-nitrophenol. The final product of 3- and 4-nitrophenol is 4-nitrocatechol.     

Reaction of p-nitrophenyl trimethylacetate with yeast carboxypeptidase. Evidence for an acyl-enzyme intermediate

The kinetics of the hydrolysis of $\text{p}$-nitrophenyl trimethylacetate catalyzed by yeast carboxypeptidase have been measured under conditions of substrate in excess and indicate that the release of $\text{p}$-nitrophenol in two discrete stages can be observed. A fast release of $\text{p}$-nitrophenol in a concentration approximating that of the enzyme is seen initially, followed by a slow release, corresponding to the “turnover” reaction of the ester. These observations provide strong support for the postulation of a three step reaction sequence including the formation and decomposition of not only a Michaelis complex but also an acyl-enzyme species.     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.     

Identification of lipolytic activity in a multitrophic population grown in wool-scour effluent

Summary A multitrophic population established in wool-scour effluent produced esterase activity with specificity to steryl, wax and triacylglyceryl esters. The wax esterase activity peaked just prior to the esterase activity toward cholesteryl oleate andp-nitrophenol palmitate. The activity was present in both the extracellular and cell membrane fractions. Partial characterization revealed the presence of multiple esterase bands on native polyacrylamide gels. The bacterial esterase activity could be enhanced by the addition of mineral salts.     

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.     

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a Bamberger rearrangement

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.     

Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated.     

Radical mechanisms of the decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct and of reactions with L-tyrosine and related compounds as studied by (15)N chemically induced dynamic nuclear polarization.

Enhanced absorption is observed in the (15)N NMR spectra of (15)NO(-)(3) during decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct at pH 5.25, indicating the formation of (15)NO(-)(3) in radical pairs [(15)NO(*)(2), HO(*)] and [(15)NO(*)(2), CO(*-)(3)]. During the reaction of peroxynitrite and the peroxynitrite-CO(2) adduct with L-tyrosine, the (15)N NMR signal of the nitration product 3-nitrotyrosine exhibits emission showing a radical pathway of its formation. The nuclear polarization is built up in radical pairs [(15)NO(*)(2), tyr(*)] generated by free radical encounters of nitrogen dioxide and tyrosinyl radicals. The (15)N NMR signal of (15)NO(-)(2) formed during reaction of peroxynitrite with L-tyrosine appears in emission. It is concluded that tyrosinyl radicals are generated by reaction of nitrogen dioxide with L-tyrosine. In contrast to this, (15)NO(-)(2) does not show (15)N chemically induced dynamic nuclear polarization (CIDNP) during reaction of the peroxynitrite-CO(2) adduct with L-tyrosine, indicating a different reaction mechanism, which is assumed to be a hydrogen transfer between CO(*-)(3) and L-tyrosine. Emission is also observed in the (15)N NMR signals of 2-nitro-4-fluorophenol, 3-nitro-4-hydroxyphenylacetic acid, 2-nitrophenol, and 4-nitrophenol during reaction of 4-fluorophenol, 4-hydroxyphenylacetic acid, and phenol with peroxynitrite and the peroxynitrite-CO(2) adduct. 3-Nitro-4-hydroxyphenylacetic acid is also observed in emission during reaction of phenylacetic acid with peroxynitrite, but is not formed with the peroxynitrite-CO(2) adduct. The magnitude of the (15)N CIDNP effect during reaction of peroxynitrite with 4-fluorophenol and of the peroxynitrite-CO(2) adduct with 4-fluorophenol and phenol is determined. It excludes the occurrence of nonradical reactions. Only weak emission signals are observed during the reaction of peroxynitrite with phenol in (15)NO(-)(2), 2-nitrophenol, and 4-nitrophenol. 2-Nitrophenol is only formed in traces, and 4-nitrophenol is only formed in higher yields. The latter might be generated in part via a nonradical pathway.     

Degradation of mixtures of phenolic compounds by <Emphasis Type="Italic">Arthrobacter chlorophenolicus</Emphasis> A6

In this study the chlorophenol-degrading actinobacterium, Arthrobacter chlorophenolicus A6, was tested for its ability to grow on mixtures of phenolic compounds. During the experiments depletion of the compounds was monitored, as were cell growth and activity. Activity assays were based on bioluminescence output from a luciferase-tagged strain. When the cells were grown on a mixture of 4-chlorophenol, 4-nitrophenol and phenol, 4-chlorophenol degradation apparently was delayed until 4-nitrophenol was almost completely depleted. Phenol was degraded more slowly than the other compounds and not until 4-nitrophenol and 4-chlorophenol were depleted, despite this being the least toxic compound of the three. A similar order of degradation was observed in non-sterile soil slurries inoculated with A. chlorophenolicus. The kinetics of degradation of the substituted phenols suggest that the preferential order of their depletion could be due to their respective pKa values and that the dissociated phenolate ions are the substrates. A mutant strain (T99), with a disrupted hydroxyquinol dioxygenase gene in the previously described 4-chlorophenol degradation gene cluster, was also studied for its ability to grow on the different phenols. The mutant strain was able to grow on phenol, but not on either of the substituted phenols, suggesting a different catabolic pathway for the degradation of phenol by this microorganism.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

Characterization of <Emphasis Type="Italic">Rhodococcus wratislaviensis</Emphasis> strain J3 that degrades 4-nitrocatechol and other nitroaromatic compounds

The bacterial strain J3 was isolated from soil by selective enrichment on mineral medium containing 4-nitrocatechol as the sole carbon and energy source. This strain was identified as Rhodococcus wratislaviensis on the basis of morphology, biochemical, physiological and chemotaxonomic characterization and complete sequencing of the 16S rDNA gene. The isolated bacterium could utilize 4-nitrocatechol, 3-nitrophenol and 5-nitroguaiacol as sole carbon and energy sources. Stoichiometric release of nitrites was measured during degradation of 4-nitrocatechol both in growing cultures and for stationary phase cells. The J3 strain was unable to degrade 4-nitroguaiacol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrobenzoic acid, 4,5-dimethoxy-2-nitrobenzoic acid and 2,3-difluoro-6-nitrophenol. The J3 strain is deposited in the Czech Collection of Microorganisms as CCM 4930.     

Is histidine involved in the catalytic mechanism of unspecific carboxylesterases?

The reaction between the liver carboxylesterases from ox and pig and the inhibitor α-bromoacetophenone was studied by amino acid analysis. A significant modification of histidine in pig liver esterase was not found, but there was a slight loss of some other residues. In ox liver esterase the total inhibition correlated with the loss of about 1.7 histidine residues. However, in contrast to previous results with chicken and ox esterases the specific active-site-directed inhibitor E 600 did not prevent the modification of the reactive histidine. It is concluded that an earlier report on the involvement of histidine in the action of liver esterases (1) is partly incorrect or perhaps applicable only to chicken liver esterase.     

Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.     

N-Butyl-N-methyl-4-nitrophenyl carbamate as a specific active site titrator of bile-salt-dependent lipases

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile-salt-dependent lipase (cholesterol esterase) was examined. N-isopropyl-O-phenyl, N-methyl-O-phenyl, N-butyl-(4-nitrophenyl), N-phenyl-(4-nitrophenyl), N-butyl-N-methyl and N-pentyl-O-phenyl carbamates were inhibitors of the enzyme activity, while O-isopropyl-N-phenyl, O-methyl-N-phenyl, O-benzyl-N-isopropyl and O-cyclohexyl-N-phenyl carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and N-butyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.     

Degradation of 4-nitroaniline by Stenotrophomonas strain HPC 135

A bacterial strain HPC 135 capable of growing on 4-nitroaniline (NA) as a source of carbon and energy was isolated from contaminated site after enrichment. Experiments revealed that the strain HPC 135 utilized 4NA as analyzed by high-performance liquid chromatography (HPLC). The presence of acetate as co-substrate did not affect the utilization of 4-nitroaniline by the isolate but cell growth was increased. Oxygen consumption studies demonstrated that strain HPC 135 could utilize various substrates such as 4-chloro-2-nitrophenol, 4-chlorobenzonitrile, 4-nitrophenol as well as 4NA. On partial 16S rDNA sequence analysis, strain HPC 135 showed highest homology with Stenotrophomonas strain based on FASTA program.     

Antimicrobial agents

In the microbial transformation of 2-chloro-4-nitrophenol and its 2-aminotridecane compound salt, 2-chloro-4-nitrophenol was found to be degraded to four metabolites only on using strains resistant to 2-chloro-4-nitrophenol. Sensitive strains lacked this property. In the case of the 2-amino-tridecane compound salt, the first reaction produced by strains resistant to 2-chloro-4-nitrophenol was dissociation of the compound salt into the two initial components, followed by degradation of 2-chloro-4-nitrophenol. Microbial degradation resulted in diminished antifungal activity. The other antimicrobi-ally active component of the compound salt, i.e. 2-aminotridecane, was not affected by biotransformation and kept its original activity.     

The catalytic properties of a proteinase isolated from sheep abomasal mucosal mast cells

The catalytic properties of a sheep mast cell proteinase (SMCP), isolated from abomasal mucosal mast cells, were investigated. The enzyme was shown to have chymotrypsin-like esterase activity, with no detectable amide activity, using a range of low molecular weight substrates. Maximal activity, against Benzyloxycarbonyl-L-tyrosine-4-nitrophenol ester, was determined to be in the range pH 7.6-8.0. Inhibitor studies showed that, unlike chymotrypsin, a serine proteinase, SMCP was found to be susceptible to the action of thiol blocking agents and chelating agents, but to be unaffected by diisopropylphosphofluoridate, a serine proteinase inhibitor.