SEARCH · PubMed Health

Results for “AMIDOHYDROLASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources

PubMed1977

New formamidase having substrate specificity for o-formylaminoacetophenone in pig liver.

The supernatant (13 000 x g, 20 min) of pig liver homogenate was filtered with Sephadex G-200 and formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9)activity in each fraction was measured. When formylkynurenine was used as substrate, two peaks of formamidase activity were observed but, with formylaminoacetophenone as substrate, only one peak was observed. Formamidase in the lower molecular weight fraction is known as kynurenine formamidase (FA I), formamidase found here in the higher molecular weight fraction has not been previously reported. This form, designated FA II has been purified about 160-fold from pig liver. The formamidase obtained has substrate specificity for o-formylaminoacetophenone only and could not hydrolyze formylkynurenine. The optimal pH was 8.5 and the Km for o-formylaminoacetophenone was 1.66-10(-3) M. This formamidase was considered to be a new enzyme and was different from FA I in molecular weight and substrate specificity. This new formamidase was present in pig, rabbit and guinea pig liver and not present in rat or mouse liver.

Acetophenones↗

PubMed1977

Coordinate and non-coordinate accululation of aspartate transcarbamylase and dihydroorotase in synchronous Chlorella cells growing on different nitrogen sources.

Regulation of the levels of aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) and dihydroorotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) was studied in synchronous cultures of the eucaryotic microorganism Chlorella. Analytical polyacrylamide gel electrophoresis and sucrose density-gradient centrifugation studies revealed that these cells contain a single aspartate transcarbamylase and a dihydroorotase with apparent molecular weights of 160 000 and 80 000, respectively. In synchronous cells cultured in nitrate medium, these two enzymes accumulated in single step-patterns over different periods of the cell cycle. In contrast, these enzymes accumulated in a coordinate manner throughout the cell cycle in ammonium medium. Experiments with inhibitors of protein and RNA synthesis indicated that dihydroorotase is stable in vivo and suggested that cell cycle changes in the turnover rate of aspartate transcarbamylase might determine whether or not these enzymes accumulate in a coordinate manner. Although uracil and uridine could be absorbed and metabolized by the cells, synthesis of these two enzymes could not be repressed by culturing synchronous cells in medium, containing high concentrations (29-40 mM) of uracil or uridine, for an entire cell cycle.

Amidohydrolases↗

PubMed1978

N-Acyl-L-aromatic amino acid deacylase in animal tissues.

An enzyme deacylating preferentially N-acyl-L-aromatic amino acids was partially purified from rat kidney. The purification procedure included DEAE-cellulose column chromatography, (NH4)2SO4 fractionation, gel-filtration on a Sephadex G-200 column and further DEAE-cellulose chromatography. The enzyme was thus separated from aminoacylase (N-acylamino-acid amidohydrolase, EC 3.5.1.14) (acylase I). Although the enzyme preparation contained other acylases, the experimental data (effect of p-chloromercuric benzoate, heat stability and inhibition between substrates) suggest that the enzyme acts preferentially on N-acyl derivatives of L-tryptophan, L-tyrosine, L-phenylalanine and L-histidine. This enzyme appears to be present in many animal tissues.

Amidohydrolases↗

PubMed1979

Pyrimidine-degrading enzymes. Purification and properties of beta-ureidopropionase of Euglena gracilis.

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, were detected in a crude extract. beta-Ureidopropionase (N-carbamoyl-beta-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of beta-ureidopropionase for beta-ureidopropionate 3.8 . 10(-5) M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60 degrees C, stimulation by Mg2+, inhibition by Cu2+, Mr approximately 1.5--2 . 10(6). beta-Ureidoisobutyrate, the intermediate of thymine degradation, and beta-ureidopropionate are competing substrates of beta-ureidopropionase (Ki = Km of beta-ureidopropionase for beta-ureidoisobutyrate 1.8 . 10(-5) M). Structural analogues of beta-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of beta-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of beta-ureidopropionase in pyrimidine degradation.

Amidohydrolases↗

PubMed1979

Kinetic mechanism of the aliphatic amidase from Pseudomonas aeruginosa.

The kinetic constants for hydrolysis and transfer (with hydroxylamine as the alternate acceptor) of the aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were determined for a variety of acetyl and propionyl derivatives. The results obtained were consistent with a ping-pong or substitution mechanism. Product inhibition, which was pH dependent, implicated an acyl-enzyme compound as a compulsory intermediate and indicated that ammonia combined additionally with the free enzyme in a dead-end manner. The uncompetitive activation of acetamide hydrolysis by hydroxylamine and the observation that the partitioning of products between acetic acid and acetohydroxamate was linearly dependent on the hydroxylamine concentration substantiated these conclusions and indicated that deacylation was at least partially rate limiting. With propionamide as the acyl donor apparently anomalous results, which included inequalities in certain kinetic constants and a hyperbolic dependence of the partition ratio on the hydroxylamine concentration, could be explained by postulating a compulsory isomerisation of the acyl-enzyme intermediate prior to the transfer reaction.

Acetamides↗

PubMed1980

Substrate specificity of penicillin amidase from E. coli.

1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.

Amidohydrolases↗

PubMed1980

Purification and characterization of a nicotinamide deamidase released into the growth medium of neuroblastoma in vitro.

Nicotinamide deamidase (nicotinamide amidohydrolase, EC 3.5.1.19) has been demonstrated in the conditioned growth medium of the M1 clonal cell line of mouse C1300 neuroblastoma. The enzyme has been purified 1200-1500-fold by Sephadex G25, hydroxyapatite, DEAE-cellulose, Sephadex G200 and NAD-Sepharose column chromatographies. The purified protein was characterized by polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions. The apparent molecular weight has been estimated to be 230,000, and the subunits had respective molecular weights of 65,000 and 50,000. Histidine was the only NH2-terminal amino acid found. The enzyme is a glycoprotein; mannose and N-acetyl-glucosamine have been identified. The effects of various ions on its activity have been investigated. The enzyme has a Km for nicotinamide in the order of 10(-6) M, a pH optimum of 7.2 and a pHi of 5.4. It is inhibited by heating and by sulfhydryl reagents. The existence of a nicotinamide deamidase with a high affinity for nicotinamide favors the operation of the Preiss-Handler pathway in M1 cells cultured in vitro. We found an induction of nicotinamide deamidase and a cellular increase of NAD with a higher nicotinamide supply and a repression of the released enzyme with supplying NAD in the nutrition medium of M1 cell cultures.

Amidohydrolases↗

PubMed1988

Aminoacylase I from hog kidney: anion effects and the pH dependence of kinetic parameters.

The hydrolysis of acetylamino acids by highly purified hog kidney aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated using flow injection analysis to determine reaction rates. We show that the distinctly bell-shaped pH versus activity profiles observed in previous studies do not reflect protonic equilibria in the enzyme, but were created by buffer effects. At low pH, anions such as phosphate, nitrate or chloride markedly increase Km. These effects are reversed at higher pH. In zwitterionic 'Good' buffers (Mes, Mops, and Bicine), maximal velocities are almost independent of pH between 6.5 and 9 for all substrates studied (Ac-LAla, Ac-LGlu, Ac-LMet, Ac-LPhe). Below pH 6.5, the catalytic constants decrease with pH, apparently due to the protonation of a carboxylate with a pKa of 5.5-6. The pH dependence of Km markedly varies among different substates. We conclude that the observed profiles all result from the dissociation of an active-site residue with a pKa of 8-8.5, which we tentatively identify as an active-site cysteine residue. A working model of aminoacylase catalysis is presented that accounts for most of the known facts.

Amidohydrolases↗

PubMed1987

Amidohydrolysis of N-methylhydantoin coupled with ATP hydrolysis.

A new enzyme, N-methylhydantoin amidohydrolase, was highly purified from Pseudomonas putida 77: it catalyzes the hydrolysis of N-methylhydantoin to N-carbamoylsarcosine with the concomitant stoichiometric cleavage of ATP to ADP and orthophosphate. The enzyme absolutely requires ATP, MG2+ and K+ for the N-methylhydantoin hydrolysis. The rapid and complete degradation of N-methylhydantoin during the cultivation of P. putida 77, which rapidly degrades creatinine via only N-methylhydantoin and which shows high activities of the enzymes involved in creatinine degradation (Yamada et al. (1985) FEMS Microbiol. Lett. 30, 337-340), seems to be due to the continuous ATP-generation during cultivation.

Adenosine Triphosphate↗

PubMed1980

N-Acetylglucosamine-asparagine levels in tissues of patients with aspartylglycosaminuria.

The levels of the main glycoprotein-derived storage compound, N-acetylglucosamine-asparagine, in various post mortem tissues of three adult patients with inherited deficiency of lysosomal 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria) were measured by gas-liquid chromatography. All aspartylglycosaminuria tissues studied contained significant amounts of N-acetylglucosamine-asparagine, whereas none of the corresponding control tissues contained detectable amounts of this compound. High levels of N-acetylglucosamine-asparagine were found in the liver (3.65 mg/g wet weight), spleen (2.24) and thyroid (2.18), and lower levels in the kidney (0.89), brain (0.53), spinal cord (0.32), sciatic nerve (0.34) and skeletal muscle (0.16). The results show that N-acetylglucosamine-asparagine accumulates chiefly in tissues with important functions in glycoprotein metabolism and/or high endocytic activity. Correlation of the results to the clinical manifestations of aspartylglycosaminuria did not reveal a direct relationship between the amount of N-acetylglucosamine-asparagine stored and the degree of organ dysfunction.

Acetylglucosamine↗

PubMed1981

Urinary sialic acid levels in aspartylglycosaminuria.

Urinary sialoglycoconjugates were studied in 22 patients with inherited deficiency of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria), in eight obligate heterozygotes, and in age- and sex-matched control subjects. Total sialic acid excretion was significantly higher in the patients (38.3 +/- 17.7 mumol/mmol creatinine, mean +/- S.D.) than in the matched controls (17.7 +/- 7.3 mumol/mmol creatinine, p less than 0.001). The sialic acid output in the heterozygotes did not differ from that of the controls. Gel filtration studies revealed that the increase in urinary sialic acid in aspartylglycosaminuria is of bound type and confined to the low molecular mass region. A linear positive correlation was found between the output of sialic acid and glycoasparagine in the individual patients (r = 0.77, p less than 0.001). The amount of sialylated metabolites excreted in urine did not correlate with the severity of clinical manifestations in aspartyl-glycosaminuria.

Adolescent↗

PubMed1987

Role of membrane N-linked oligosaccharides in host cell interaction with invasive forms of Trypanosoma cruzi.

The removal of N-linked oligosaccharides by peptide-N4-[N-acetyl-beta-glucoseaminyl]asparagine amidase (previously known as aspartoglycosylamine amidohydrolase and abbreviated N-glycanase) from the surface of blood or insect-transmissible forms of Trypanosoma cruzi markedly increased the capacity of these organisms to associate with (i.e., bind and penetrate) either mouse peritoneal macrophages or rat heart myoblasts. This effect was evidenced by a significant elevation in both the percentage of infected host cells and the average number of parasites per 100 cells. Conversely, N-glycanase treatment of either host cell markedly reduced both parameters to levels significantly below those obtained with cells mock treated with medium alone. The N-glycanase effect on the parasites was inhibited by heat inactivation of the enzyme or by the presence of fetuin, an N-glycanase substrate. The enhanced capacity of N-glycanase-treated T. cruzi to engage the host cells started to subside 2 h after the treatment, indicating the reversibility of the effect. The decreased reactivity of N-glycanase-treated macrophages or myoblasts with T. cruzi suggests that N-linked oligosaccharides on these host cells are involved in the initial phase of the cell infection process. Instead, because T. cruzi interacted more effectively with host cells after treatment with N-glycanase, parasite surface N-linked oligosaccharides would seem to interfere with the association.

Amidohydrolases↗

PubMed1982

Partial purification and characterization of N-acetylmuramyl-L-alanine amidase from human and mouse serum.

The enzyme N-acetylmuramyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) has been detected in human, mouse, rabbit, bovine and sheep sera. A method for detection of amidase activity using [14C]peptidoglycan monomer as the substrate has been developed. Partial purification of human and mouse amidase was achieved by gel chromatography on Bio-Gel A-1.5 m, DEAE-Sephadex A-50 and Sephadex G-100. Both amidase preparations exhibited maximal activity at pH 9.0 in Tris-HCl buffer and required Mg2+ for maximal activity. Following digestion of peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala, the disaccharide GlcNAc-MurNAc and the corresponding pentapeptide L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala were formed and subsequently isolated and chemically characterized. The enzyme therefore acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine. The glycan linked, peptide-not-cross-linked peptidoglycan dimer was also shown to be a substrate for human and mouse amidase.

Amidohydrolases↗

PubMed1985

Evidence for a pH-dependent isomerization of Clostridium oroticum dihydroorotase.

Analytical gel permeation chromatography on both Sephadex and polyacrylamide columns shows that Clostridium oroticum dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) undergoes a large decrease in molecular size when the pH is decreased from 8 to 6. The Stokes radius decreases from about 40 A to 36 A. Neither the molecular size nor kinetic properties are dependent on protein concentration. Thus, the decreased molecular size reflects a pH dependent isomerization of the enzyme.

Amidohydrolases↗

PubMed1988

The turnover of lysosomal glycosylasparaginase in rat liver.

Rat liver glycosylasparaginase (N4-(beta-N-acetyl-D-glucosaminyl)-L-asparagine amidohydrolase (EC 3.5.1.26) was irreversibly inhibited in vitro by the asparagine analogue 5-diazo-4-oxo-L-norvaline (DONV). Following the abolition of this lysosomal hydrolase by DONV in rats, the enzyme activity in liver recovered exponentially. Assuming a zero order rate of protein synthesis, the half-life of the glycosylasparaginase in rat liver was measured to be 2 days.

Amidohydrolases↗

PubMed1990

Purification and characterization of N-acetylmuramoyl-L-alanine amidase from human serum.

Purification to homogeneity of the N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120,000-130,000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of Mr = 125,000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of Mr = 57,000 and another of Mr = 70,000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protein was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurrence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Escherichia coli or Bacillus cereus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbial muramoyl amidases.

Amidohydrolases↗

PubMedDate not supplied

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa.

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.

Acetamides↗

PubMed1991

Pyrimidine catabolism in Pseudomonas aeruginosa.

Pyrimidine catabolism in Pseudomonas aeruginosa was investigated. It was found that the pyrimidine bases uracil and thymidine as well as their respective reductive catabolic products could be utilized as sole sources of nitrogen. Reductive degradation of the pyrimidine bases was noted. The reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase were all detected in minimal medium grown cells. Induction of pyrimidine catabolism by uracil was observed in this pseudomonad. Pyrimidine degradation in P. aeruginosa was not subject to catabolite repression.

Amidohydrolases↗