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)

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.
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PMID:Immobilization-stabilization of penicillin G acylase from Escherichia coli. 209 28

Aflatoxin B1 (AFB1) was shown to react primarily with one or more lysine residues in serum albumin (SA), accounting for more than half of the total binding to this protein. The radioactivity associated with SA following administration of [U-14C]AFB1 to rats was cleared with a half-life of 2.5 days, which is not significantly different from the half-life of unmodified albumin in the normal rat. The product isolated from a Pronase digest of in vivo-modified SA was identical by chromatographic retention time and u.v. and mass spectroscopy to the synthetic product obtained by the acylase-catalyzed deacetylation of the reaction product of N alpha-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB1. The latter was characterized by u.v., fluorescence, 500 MHz 1H-n.m.r. and fast atom bombardment mass spectrometry. The spectral data strongly support a structure in which the terminal dihydrofuran ring of AFB1 has been converted to a pyrrolinone ring. It is proposed that the initial adduct is formed by condensation of the dialdehyde tautomer of 8,9-dihydro-8,9-dihydroxy-AFB1, with the epsilon-amino group of lysine, to form a Schiff base, and that the Schiff base undergoes an Amadori rearrangement to an alpha-amino ketone. The pyrrolinone ring is formed by condensation of the amino group with the remaining aldehyde to yield the final product. The purified product was relatively stable but was shown to decompose significantly under the conditions used to isolate it from modified SA.
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PMID:Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats. 311 39

1. Pseudomonas sp. N.C.I.B. 8858 grew well on d- and l-1-aminopropan-2-ol and on aminoacetone. 2. Cell-free extracts possessed high activities of inducibly formed l-1-aminopropan-2-ol-NAD(+) oxidoreductase, amino alcohol-ATP phosphotransferase, dl-1-aminopropan-2-ol O-phosphate phospho-lyase and aldehyde-NAD(+) oxidoreductase, but no 1-aminopropan-2-ol racemase or d-1-aminopropan-2-ol-NAD(+) oxidoreductase. 3. The amino alcohol kinase (activated by ADP) was non-stereospecific towards 1-aminopropan-2-ol and was one-third as active with ethanolamine. The phospho-lyase was active with l- and d-1-aminopropan-2-ol O-phosphate, but ethanolamine O-phosphate was only one-tenth as active as its higher homologues. The purified aldehyde dehydrogenase was active with propionaldehyde, acetaldehyde and also with methylglyoxal. The previously observed 2-oxo aldehyde dehydrogenase activity was considered to be due to the broadly specific aldehyde dehydrogenase. 4. Mutants of Pseudomonas sp. N.C.I.B. 8858 deficient in 1-aminopropan-2-ol kinase, 1-aminopropan-2-ol O-phosphate phospho-lyase, aldehyde dehydrogenase or an enzyme involved in propionate metabolism were incapable of growth on aminoacetone or 1-aminopropan-2-ol as carbon source, although all except the kinase- or phospho-lyasedeficient mutants could use these compounds and ethanolamine as nitrogen sources. The aldehyde dehydrogenase-deficient mutants produced copious amounts of propionaldehyde and acetaldehyde during growth on the corresponding amino alcohols. 5. The path of aminoacetone metabolism in Pseudomonas sp. N.C.I.B. 8858 was concluded to involve l-1-aminopropan-2-ol, the O-phosphate ester of this compound, propionaldehyde and propionate as obligatory intermediates. d-1-Aminopropan-2-ol was metabolized by the same route as the l-isomer, gratuitously inducing formation of the stereospecific l-1-aminopropan-2-ol dehydrogenase. 6. Extracts of the pseudomonad grown with ethanolamine as the nitrogen source were devoid of 1-aminopropan-2-ol dehydrogenase, the kinase and the phospho-lyase, but exhibited cobamide coenzyme-dependent deaminase activity. Mutants deficient in kinase or phospho-lyase (deaminating) grew well on ethanolamine as the nitrogen source. Ethanolamine deaminase was inactive with, but inhibited by, 1-aminopropan-2-ol.
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PMID:Microbial metabolism of amino alcohols. Aminoacetone metabolism via 1-aminopropan-2-ol in Pseudomonas sp. N.C.I.B. 8858. 436 43

The paper describes a method to follow acylase activity. The method is based on spectrophotometry of the amino acid released, using o-phthalic aldehyde and mercaptoethanol. The major advantage of the method are its high sensitivity and speed. With the aid of the method kinetic parameters of hydrolysis of N-acetyl-L-methionine and N-acetyl-D,L-methionine catalyzed by pig kidney acylase were determined. Michaelis constants at pH 7.5 were estimated to be 5 +/- 1 and 10 +/- 2, respectively; this being in consistency with the data in the literature. It was shown that N-acetyl-D-methionine, acetate ion and methionine at the concentrations tested (0.01-0.05 M) did not inhibit acylase from a pig kidney.
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PMID:[Method of measuring activity of amino acid acylase]. 738 16

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.
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PMID:Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine). 1136 56

Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20 degrees C, and immobilization time of 120 min. Unreacted aldehyde groups were quenched by reaction with a low-molecular-weight material such as L-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.
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PMID:Immobilization of glutaryl-7-aminocephalosporanic acid acylase on silica gel and enhancement of its stability. 1266 70

A very simple strategy, based on the intermolecular cross-linking of associated proteins by using aldehyde-dextrans, has been proposed to detect protein-protein interactions. Aldehyde-dextran was able to cross-link different enzymes composed of several polypeptide chains (e.g., trypsin and penicillin G acylase), proteolyzated proteins (e.g., extracts from porcine pancreas) and finally, an immunocomplex (horseradish peroxidase/anti-horseradish peroxidase). This cross-linked immunocomplex could be selectively adsorbed on immobilized anti-rabbit IgG. The presence of unspecific covalent attachment between unrelated protein molecules was not detected. Thus, this strategy permits the cross-linking of different protein components and avoids the formation of nonspecific protein-protein associations.
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PMID:Determination of protein-protein interactions through aldehyde-dextran intermolecular cross-linking. 1535 35

Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.
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PMID:Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool. 1717 52

The acylase from Arthrobacter viscosus was immobilized, studied in the enzymatic synthesis of some cephalosporins by kinetically controlled N-acylation (kcNa) of different cephem nuclei, and compared with the penicillin G acylase (PGA) from Escherichia coli. The reaction outcomes were dependent on the acylase microbial source and on the type of immobilization support. Generally, both enzymes, when immobilized onto hydrophilic resins such as glyoxyl-agarose (activated with aldehyde groups), displayed higher synthetic performances in comparison with hydrophobic acrylic epoxy-supports like Eupergit C. The kcNa of 7-amino cephalosporanic acid catalyzed by A. viscosus immobilized on glyoxyl-agarose afforded a quantitative conversion in 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Delta(3)-cephem-4-carboxylic acid, a useful intermediate for the synthesis of Cefamandole and Cefonicid. Similar results were obtained in the synthesis of these cephalosporins by direct acylation of the corresponding 3'-functionalized nucleus. In these reactions, A. viscosus displayed higher synthetic performances than the PGA from E. coli.
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PMID:Enzymatic synthesis of cephalosporins. The immobilized acylase from Arthrobacter viscosus: a new useful biocatalyst. 1787 93

We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].
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PMID:Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila. 1861 49


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