Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39-45 degrees C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30 degrees C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg2+, EDTA, D-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to L-enantiomers in the C-terminal position.
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PMID:Purification and characterization of a newly screened microbial peptide amidase. 859 40

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.
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PMID:Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. 861 77

The CHA1 gene of Saccharomyces cerevisiae encodes the catabolic L-serine (L-threonine) deaminase responsible for the utilization of serine/threonine as nitrogen sources. Previously, we identified two serine/threonine response elements in the CHA1 promoter, UASCHA. We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHA1 transcription. The cha4-1 allele causes noninducibility of a CHA1 p-lacZ translational gene fusion, indicating that Cha4p exerts its action through the CHA1 promoter. Molecular and genetic mapping positioned the cha4 locus 17 cM centromere proximal to put1 on chromosome XII. The coding region of CHA4 predicts a 648-amino acid protein with a DNA-binding motif (residues 43-70) belonging to the Cys6 zinc cluster class. Gel retardation employing a recombinant peptide, Cha4p1-174, demonstrated that the peptide in vitro specifically binds UASCHA. Binding is abolished by a G-C to T-A mutation in the middle bases of the two CEZ-elements in UASCHA. The transcriptional activating ability of UASCHA derivatives in vivo correlates with their ability to bind Cha4p1-174 in vitro. We conclude that Cha4p is a positive regulator of CHA1 transcription and that Cha4p alone, or as part of a complex, is binding UASCHA.
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PMID:Cha4p of Saccharomyces cerevisiae activates transcription via serine/threonine response elements. 888 13

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.
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PMID:Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase. 907 Feb 24

1-Aminocyclopropane-1-carboxylate (ACC) deaminase catalyzes the cyclopropane ring fragmentation and deamination of ACC. Replacement of cysteine with alanine at a reactive thiol site, Cys-162, of ACC deaminase did not affect the enzyme activity, in spite of the previous result that modification of Cys-162 caused complete loss of the enzyme activity. Substitution of glycine or valine for the cysteine residue gave a higher Km for ACC without a significant change of the K0, indicating that changes of the amino acid side chain had structural effects on substrate binding. Replacement of lysine with alanine at the pyridoxal phosphate (PLP) binding site of the ACC deaminase caused a lower content of PLP and loss of detectable activity of ACC deamination. This mutant enzyme, K51A, showed absorption peaks at 330 nm and 405 nm. The peak at 405 nm was shifted to about 425 nm by the addition of ACC, D-, L-alanine, and D-, L-serine. The formation of aldimine complexes indicated by the spectral shift was reversible. It is suggested that lysine 51 affects the formation of holoenzyme and is important in catalysis.
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PMID:Substitutions of alanine for cysteine at a reactive thiol site and for lysine at a pyridoxal phosphate binding site of 1-aminocyclopropane-1-carboxylate deaminase. 909 53

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 --> Asn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.
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PMID:Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes. 934 49

Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing. The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination. Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins. However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed. In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine. These may be non-competent DM domains able to bind but not act upon their substrate. Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch. The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes. Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates.
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PMID:Statistical modelling and phylogenetic analysis of a deaminase domain. 954 71

Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro-PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p-nitrophenyl-p'-guanidinobenzoate HCl and diisopropylflurophosphate. The NH2-terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF-I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF-II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor.
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PMID:In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. 965 93

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.
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PMID:Synthesis and characterization of diazomethylarachidonyl ketone: an irreversible inhibitor of N-arachidonylethanolamine amidohydrolase. 965 59

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.
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PMID:Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions. 980 66


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