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At least 19 recordsLinked to original sources
PubMed1988

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex.

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released (14)CO(2) from [ureido-(14)C]ureidoglycolate under conditions in which urease is not detectable. CO(2) and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K(m) of 85 micromolar for ureidoglycolate. Glyoxylate and CO(2) are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO(2) and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2'-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [(14)C]urea production from [2,7-(14)C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of (14)CO(2) release from [2,7-(14)C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of (14)CO(2) is not dependent on the accumulation of free intermediates. That [2,7-(14)C]allantoate dependent (14)CO(2) release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO(2) without a urea intermediate.

Journal Article↗
PubMed2004

A novel N-carbamoyl-L-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-L-cysteine amidohydrolase expressed in Escherichia coli.

N-carbamoyl-L-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50 degrees, respectively. The enzyme required Mn(2+) ion for activity expression and was inhibited by EDTA, Hg(2+) and sulfhydryl reagents. The enzyme was strictly specific for the L-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-L-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel L-carbamoylase, different from the known L-carbamoylases.

Amidohydrolases↗
PubMed2000

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Amidohydrolases↗
PubMed1995

Purification and characterization of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas putida 77.

N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300,000. It is a tetramer of two identical small subunits (M(r) 70,000) and two identical large subunits (M(r) 80,000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The Km and Vmax values for N-methylhydantoin were 32 microM and 9.0 mumol.min-1.mg protein-1. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, DL-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin. 2-Pyrrolidone, 2-oxazolidone, delta-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, D-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis.

Adenosine Triphosphate↗
PubMed1998

Synthesis and characterization of diazomethylarachidonyl ketone: an irreversible inhibitor of N-arachidonylethanolamine amidohydrolase.

N-Arachidonylethanolamine (AEA), a putative endogenous agonist of neuronal (CB1) cannabinoid receptors, is a substrate for N-arachidonylethanolamine amidohydrolase (AEA amidohydrolase), a serine amidase present in cell membranes. Following a strategy that has been used to develop inhibitors that covalently bind to the active site of serine peptidases, diazomethyl arachidonyl ketone (DAK) was synthesized and its effects on AEA amidohydrolase were determined. DAK inhibits the hydrolysis of AEA by rat brain membranes with an IC50 value of 0.5 microM. At low concentrations, DAK reduces the Vmax and increases the K(m) of the enzyme for its substrate AEA, which suggests that it is both a competitive and noncompetitive inhibitor. At higher concentrations, DAK inhibition is completely noncompetitive. DAK inhibition of membrane-associated AEA amidohydrolase is irreversible because hydrolytic activity is not restored with extensive washing or dialysis of the membranes. Furthermore, DAK inhibition is not reversible by anion exchange chromatography of the subsequently solubilized enzyme. In contrast, DAK inhibition of detergent-solubilized enzyme exhibits competitive kinetics and is reversible upon ion exchange chromatography. Exposure of C6 glioma cells to DAK results in concentration-related inhibition of AEA amidohydrolase activity in cellular membranes with an IC50 value of 0.3 microM. In summary, these studies demonstrate that DAK is an irreversible inhibitor of AEA amidohydrolase in its native membrane and provides a useful tool with which to study the role of AEA amidohydrolase in the termination of action of AEA.

Amidohydrolases↗
PubMed1993

Synthesis of beta-lactam antibiotics containing alpha-aminophenylacetyl group in the acyl moiety catalyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase.

D-(-)-Phenylglycyl-beta-lactamide amidohydrolase was isolated from Xanthomonas sp., purified, and characterized. A characteristic feature of the enzyme is its high specificity for substrates containing an alpha-aminophenylacetic group in the acyl moiety. Cephalexin and D-C-(-)-phenylglycine methyl ester (MEPG), being nonspecific penicillin acylase (EC 3.5.1.11) substrates, have the highest values of bimolecular constant kcat/Km (2.8 x 10(5) and 2.0 x 10(5) M-1 x s-1, respectively) in the case of amidohydrolase. On the contrary, benzylpenicillin is not hydrolyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase. The other peculiarity of the enzyme is its affinity for the charged forms of substrates. Using the amidohydrolase, it was found that the values of delta Go'pH7.0 for hydrolysis of the amide bond in cephalexin and ampicillin are -3.3 and -2.3 kJ mol-1, respectively. They are less by a minimum of 2.7 kJ mol-1 than those for other beta-lactam antibiotics. Detailed thermodynamic and kinetic studies of the synthesis of cephalexin from MEPG and 7-aminodeacetoxycephalosporanic acid (7-ADCA) catalyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase were undertaken. A kinetic scheme is proposed which describes well the experimental curves. The value of conversion of "nucleus" was found to be 76% when the synthesis was carried out from a 31.5 mM solution of 7-ADCA and an 88.5 mM solution of MEPG at pH 6.2 (optimum conditions). A 75% conversion of 7-aminocephalosporanic acid (7-ACA) was achieved in the synthesis of cephaloglycine catalyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase.

Amidohydrolases↗
PubMed1997

Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase.

Mammalian brain as well as mouse neuroblastoma (N18TG2) and rat basophilic leukaemia (RBL) cells were previously shown to contain "anandamide amidohydrolase', a membrane-bound enzyme sensitive to serine and cysteine protease inhibitors and catalyzing the hydrolysis of the endogenous cannabimimetic metabolite, anandamide (arachidonoyl-ethanolamide). With the aim of developing novel inhibitors of this enzyme, we synthesized three arachidonic acid (AA) analogues, i.e. arachidonoyl-diazo-methyl-ketone (ADMK), ara-chidonoyl-chloro-methyl-ketone (ACMK) and O-acetyl-arachidonoyl-hydroxamate (AcAHA), by adding to the fatty acid moiety three functional groups previously used to synthesize irreversible inhibitors of serine and cysteine proteases. The three compounds were purified and characterized by proton nuclear magnetic resonance and electron impact mass spectrometry. Their effect was tested on anandamide amidohydrolase partially purified from N18TG2 and RBL-1 cells and porcine brain. Pre-treatment of the enzyme with each compound produced a significant inhibition, with ADMK being the most potent (IC50 = 3, 2 and 6 microM) and AcAHA the weakest (IC50 = 34, 15 and 25 microM) inhibitors. The inactivated enzyme regained its full activity when chromatographed by anion-exchange chromatography, suggesting that none of the compounds inhibited the amidohydrolase in a covalent manner. Accordingly, Lineweaver-Burk profiles showed competitive inhibition by each compound. Conversely, the irreversible inhibitor of cytosolic phospholipase As, methyl-arachidonoyl-fluoro-phosphonate (MAFP), covalently inhibited the amidohydrolase. MAFP was active at concentrations 10(3) times lower than those reported for phospholipase A2 inhibition, and is the most potent anandamide amidohydrolase inhibitor so far described (IC50 = 1-3 nM). MAFP, ADMK and ACMK, probably by inhibiting anandamide degradation, produced an apparent increase of the in vitro formation of anandamide from its biosynthetic precursor N-arachidonoyl-phosphatidyl-ethanolamine.

Amidohydrolases↗
PubMed1995

Anandamide amidohydrolase activity in rat brain microsomes. Identification and partial characterization.

An amidohydrolase activity present in rat brain microsomes catalyzes the hydrolysis of N-arachidonoyl-[3H]ethanolamine ([3H]anandamide), an endogenous cannabimimetic substance, forming [3H]ethanolamine and arachidonic acid. Amidohydrolase activity is maximal at pH 6 and 8, is independent of divalent cations, has an apparent Km for [3H]anandamide of 12.7 +/- 1.8 microM, and has a Vmax of 5630 +/- 200 pmol/min/mg of protein. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, and p-bromophenacyl bromide, a histidine-alkylating reagent, inhibit the activity, whereas N-ethylmaleimide and various nonselective peptidase inhibitors (EDTA, o-phenanthroline, bacitracin) have no effect. Brain amidohydrolase activity exhibits high substrate specificity for [3H]anandamide; N-gamma-linolenoyl-, N-homo-gamma-linolenoyl-, and N-11,14-eicosadienoyl- are hydrolyzed at markedly slower rates. Moreover, N-11-eicosaenoyl- and N-palmitoyl-[3H]ethanolamine are not hydrolyzed. [3H]Anandamide hydrolysis is inhibited competitively by nonradioactive anandamide and by other N-acylethanolamines with the following rank order of potency: anandamide > N-linoleoyl- = N-cis-linolenoyl- = N-gamma-linolenoyl- = N- homo-gamma-linolenoyl- > N-11,14-eicosadienoyl- > N-oleoyl- > N- docosahexaenoyl- > N-docosatetraenoyl > N-linoelaidoyl- > N-eicosaenoyl- > N- palmitoyl > or = N-elaidoyl- = N-eicosanoyl-ethanolamine = no effect. Amidohydrolase activity is high in liver and brain and low in heart, kidney, intestine, stomach, lung, spleen, and skeletal muscle. Within the central nervous system, highest activity is found in globus pallidus and hippocampus, two regions rich in cannabinoid receptors, and lowest activity is found in brainstem and medulla, where cannabinoid receptors are sparse. The results, showing that brain amidohydrolase activity is selective for anandamide and enriched in areas of the central nervous system with high density of cannabinoid receptors, suggest that this activity may participate in the inactivation of anandamide at its sites of action.

Amidohydrolases↗
PubMed1991

4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme.

4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V(max) and a four-fold higher K(m) value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components.

Journal Article↗
PubMed1994

Thermostable N-carbamoyl-D-amino acid amidohydrolase: screening, purification and characterization.

A thermostable N-carbamoyl-D-amino acid amidohydrolase was found in the cells of newly isolated bacterium. Blastobacter sp. A17p-4. The bacterium also showed D-specific hydantoinase activity. The N-carbamoyl-D-amino acid amidohydrolase activity of the cells exhibited a temperature optimum at 50-55 degrees C, and was stable up to 50 degrees C. The N-carbamoyl-D-amino acid amidohydrolase of Blastobacter sp. A17p-4 was purified to homogeneity and characterized. It has a relative molecular weight of about 120,000 and consists of three identical subunits with a relative molecular weight of about 40,000. N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme. It has been suggested that D-amino acid production from DL-5-substituted hydantoin involves the action of a series of enzymes involved in pyrimidine degradation, namely amide-ring opening enzyme, dihydropyrimidinase, and N-carbamoylamide hydrolyzing enzyme, beta-ureidopropionase. However, the purified enzyme did not hydrolyze beta-ureidopropionate; suggesting that the N-carbamoyl-D-amino acid amidohydrolase coexisting with D-specific hydantoinase, probably dihydropyrimidinase, in Blastobacter sp. A17p-4 is different from beta-ureidopropionase.

Amidohydrolases↗
PubMed2001

Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.

Amidohydrolases↗
PubMed1995

Primary structure of N-acyl-D-glutamate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.

The gene coding the N-acyl-D-glutamate amidohydrolase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete DNA sequence was determined. The N-acyl-D-glutamate amidohydrolase structural gene consists of 1,464 nucleotides and encodes 488 amino acid residues. The molecular weight of the enzyme was calculated to be 51,490. This value is close to the apparent molecular weight of 59,000 determined for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence of the recombinant protein exactly matches the amino acid sequence derived from the DNA sequence and that determined from the Alcaligenes A-6 enzyme (NH2-MQEKLDLVIEGGWVIDGLGG). The deduced amino acid sequence of the cloned N-acyl-D-glutamate amidohydrolase showed high sequence homology with those of N-acyl-D-aspartate amidohydrolase (46%) and D-aminoacylase (47%) from Alcaligenes A-6. This fact strongly suggests that these three enzymes have evolved from a common ancestral gene.

Alcaligenes↗
PubMed1979

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.

Amidohydrolases↗
PubMed2002

Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis.

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.

Arabidopsis↗
PubMed1986

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties.

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding beta-ureido acids. Previous work has shown that incubation of this Zn2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226, 469-483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid greater than o-phenanthroline greater than 2,6-dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is well-described by ionization of a single group with a pK of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both Vmax and the apparent affinity for substrate. Conversely, binding of substrate decreases the pK of this group to about 5. L-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of L-dihydroorotic acid increases the pK of the ionizable group to 6.5. The agreement between the pK in the enzyme-L-dihydroorotic acid complex and the higher pK observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of acid, and chelators. The different effects of substrate and L-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.

Amidohydrolases↗
PubMed2001

Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization.

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K(m) of 3.7 microM and V(max) 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9-10, was abolished by phenylmethylsulfonyl fluoride and was Ca(2+)- and Mg(2+)-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.

Amidohydrolases↗
PubMed2003

N-Morpholino- and N-diethyl-analogues of palmitoylethanolamide increase the sensitivity of transfected human vanilloid receptors to activation by anandamide without affecting fatty acid amidohydrolase activity.

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca(2+) influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH(2)CH(2)OH group was replaced by N-CH(2)CH(2)Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH(2))(12)CH(3)] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH(2)CH(3))(2) (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 microM AEA to a greater degree than the parent compound, whereas the N-(CH(3))(2) analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca(2+) influx into hVR1-HEK293 cells. EC(50) values for AEA to produce Ca(2+) influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 microM in the presence of 0, 1, 3 and 10 microM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 microM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH(2)CH(3) and N-CH(CH(3))(2) analogues (22 and 23, respectively). 22 and 23 produced a small ( approximately 25%) inhibition of the binding of [(3)H]-CP55,940 and [(3)H]-WIN 55,212-2 to CB(1) and CB(2) receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 microM [(3)H]-AEA by rat brain fatty acid amidohydrolase with IC(50) values of 5.6 and 11 microM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 microM [(3)H]-AEA into C6 glioma cells were seen at 10 microM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.

Amides↗
PubMed2007

An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme.

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells acting through deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones. In addition, both eukaryotic HDACs as well as their bacterial counterparts were reported to also act on non-histone targets. However, we are still far from a comprehensive understanding of the biological activities of this ancient class of enzymes. In the present paper, we studied in more detail the esterase activity of HDACs, focussing on the HDAH (histone deacetylase-like amidohydrolase) from Bordetella/Alcaligenes strain FB188. This enzyme was classified as a class 2 HDAC based on sequence comparison as well as functional data. Using chromogenic and fluorogenic ester substrates we show that HDACs such as FB188 HDAH indeed have esterase activity that is comparable with those of known esterases. Similar results were obtained for human HDAC1, 3 and 8. Standard HDAC inhibitors were able to block both activities with similar IC(50) values. Interestingly, HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) also showed inhibitory activity against porcine liver esterase and Pseudomonas fluorescens lipase. The esterase and the amidohydrolase activity of FB188 HDAH both appear to have the same substrate specificity concerning the acyl moiety. Interestingly, a Y312F mutation in the active site of HDAH obstructed amidohydrolase activity but significantly improved esterase activity, indicating subtle differences in the mechanism of both catalytic activities. Our results suggest that, in principle, HDACs may have other biological roles besides acting as protein deacetylases. Furthermore, data on HDAC inhibitors affecting known esterases indicate that these molecules, which are currently among the most promising drug candidates in cancer therapy, may have a broader target profile requiring further exploration.

Alcaligenes↗