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 thermophilic bacillus growing on acetamide as both carbon and nitrogen sources produces an inducible amidase. This amidase hydrolysed the following amides in decreasing order or activity, in comparison with acetamide (1.00): propionamide (0.97), fluoroacetamide (0.84), formamide (0.35) and glycinamide (0.12). Cyanoacetamide, dimethylacetamide, dimethylformamide and urea also induced the synthesis of the amidase, but were not substrates of the enzyme. Studies with protoplasts suggest that the amidase is located in the cytoplasm. Glucose strongly inhibited amidase synthesis; and limiting nitrogen did not release this inhibition. Urea strongly inhibited amidase activity in a competitive manner; but the inhibition caused by iodoacetamide and cyanoacetamide was non-competitive. Both thioacetamide and thiourea were effective inhibitors of enzyme induction. Bacteria grown on a succinate-minimal medium exhibited a lag in amidase synthesis, which could be eliminated by decreasing the concentration of succinate. Acetate- or pyruvate-grown cultures behaved similarly, while those grown on alanine or glutamate exhibited no lag in enzyme induction. In the mutant strain E21, repression of amidase synthesis by glucose was much less evident and no lag for induction was apparent with any of the other carbon sources mentioned.
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PMID:Regulatory properties of an inducible aliphatic amidase in a thermophilic bacillus. 93 86

The N,N-dimethylformamide-hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49-fold purification, a 24% yield and a final specific activity of 1.98 mumol N,N-dimethylformamide (DMF) hydrolyzed min-1 (mg protein)-1. The native DMFase has a relative molecular mass of 250 000 and is composed of two light-chain (Mr = 15 000) and two heavy-chain (Mr = 105 000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20 degrees C. The activity of the enzyme is inhibited by metal-chelating agents such as EDTA and 2,2'-dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron-containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm. DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40 degrees C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short-chain aliphatic amides such as DMF, N-ethylformamide and N-methylformamide. N,N-dimethylformamide, N,N-dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis-Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver-Burk plot.
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PMID:Purification and characterization of N,N-dimethylformamidase from Pseudomonas DMF 3/3. 373 81

The kinetic parameters Km and kcat and the resulting proteolytic coefficients kcat/Km for the hydrolysis of blocked alanine peptide esters (X(Ala)nOMe) and -p-nitroanilides (X(Ala)n-pNA) of variable length (n = 1 to 5 alanine residues) by the cationic, microbial serine protease thermitase are determined in order to delineate the number of subsites involved in catalysis. Thermitase has at least five secondary subsites (S1 to S5) being hydrophobic in S1 to S4. Arrhenius plots for both, esterase and amidase activity were biphasic with a break at 30 degrees C, followed by a downward bend. The influence of dimethylformamide, solute for many substrates, on the thermitase-catalyzed esterolysis of Z(Ala)2OMe was also investigated. In contrast to the kcat values being unaffected by 5 to 30% dimethylformamide, the Km values increased logarithmically with enhancing its concentration.
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PMID:[The kinetics of hydrolysis of alanine peptide esters and -p-nitroanilides by thermitase, a thermostable serine protease from Thermoactinomyces vulgaris: secondary specificity, influence of temperature and solute]. 389 Aug 45

An extracellular protease has been isolated and partially purified from the extreme halophile Halobacterium halobium (ATCC 43214). The major enzyme component has a M(r) of 66,000 and is highly dependent upon salt concentrations near saturation for catalytic activity and stability. In aqueous solutions, a decrease in the NaCl concentration from 4 to 1 M results in an increase of nearly three orders of magnitude in the first-order rate constant of inactivation at 30 degrees C. Salt effects the stability of the enzyme in a cooperative manner, with a Hill coefficient of 4.1, which is similar to that of other enzymes from extreme halophiles. The enzyme activity is dramatically affected by the salt concentration, with a loss of 2.5 orders of magnitude in kcat/Km in going from 4 to 0 M NaCl. This loss in catalytic efficiency is primarily due to a dramatic increase in the Km for the substrate in low-salt media. Thermodynamic analysis revealed that this Km increase was mainly the result of increased solubility of the synthetic peptide substrate in low-salt media, which dramatically increases the ground-state stability of the substrate. This results in an effectively reduced substrate partitioning from the bulk solution into the enzyme's active site and an increased value of Km. The halophilic protease is also active in DMF/water mixtures, albeit with novel catalytic properties. In 33% (v/v) DMF in aqueous buffer, the esterase activity of the enzyme is ca. 80-fold higher than the corresponding amidase activity. This contrasts to the situation in pure aqueous buffer, in which the esterase activity is only fourfold higher than the amidase activity. The increased esterase activity relative to amidase activity prompted us to investigate the use of the protease in kinetically controlled peptide synthesis. The enzyme has a broad acyl donor substrate specificity and can effectively use amino acid esters of Phe, Tyr, Trp, Ser, Gly, and Ala. The enzyme is significantly more selective for the amino acid amide, preferring Gly in the P'1 site. A series of glycine-containing oligopeptides have been prepared in yields up to 76% without degradation due to secondary hydrolysis.
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PMID:Catalytic properties and potential of an extracellular protease from an extreme halophile. 776 32

Parameters relevant to the thermodynamically controlled synthesis of cephalothin utilizing highly active stabilized penicillin G acylase derivatives were studied. These included solubility/stability of substrates, enzyme derivative activity/stability, reaction course and synthetic yields. These parameters were altered by varying the pH, dimethylformamide concentration and temperature. Simultaneous optimization of the selected parameters could not be achieved with a single set of conditions. However, continuous adjustment of conditions throughout the reaction course allowed each parameter to be optimized (dynamic reaction design). This strategy works by optimizing those parameters that are critical to the overall reaction at a given point, whilst leaving others sub-optimal when their contribution to the total is minimal. This strategy has achieved a 90% transformation of antibiotic nucleus to cephalothin at a final concentration of 20 g/l, high enzyme and reactant stability, with a reaction period of 3 h (using 1 ml of derivative/40 ml of reaction solution).
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PMID:Dynamic reaction design of enzymic biotransformations in organic media: equilibrium-controlled synthesis of antibiotics by penicillin G acylase. 886 5

Catalytic properties and conformational stability of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) were studied in water-N,N-dimethylformamide (DMF) and water-dioxane solvent mixtures. Beside the prompt inhibition the solvents caused further inactivation during incubations. In the presence of 5% DMF content the inactivation proceeds with a well-measurable rate (t1/2 39 min), while in the case of 20% DMF the enzyme practically lost its starting activity during 50 min incubation (t1/2 13 min). The K(m) value of the enzyme increased about three times with increasing DMF concentrations up to about 2.6 M DMF, while the Vmax value decreased practically to zero in the same concentration range.
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PMID:Studies on kinetic parameters and stability of aminoacylase in non-conventional media. 986 60

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two alpha- and two beta-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.
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PMID:Cloning and expression of the N,N-dimethylformamidase gene from Alcaligenes sp. strain KUFA-1. 1066 42

Peptide amidase-catalysed amidations of the C- terminal carboxylic group of peptides were studied using model substrates of a large series of N(alpha)-protected di-, tri-, tetra- and penta-peptides in the presence of NH4HCO3 as the ammonium source. The maximal yields of amide syntheses were achieved in a medium consisting of acetonitrile with 20-25 vol% of dimethylformamide and 3 vol% of water. Under these conditions, the substrate specificity of the enzyme was more restricted in the synthetic reaction than was found for the amide hydrolysis. Elongation of the peptide chain had a negative effect on enzymic amidation. Thus the direct amidation of N(alpha)-t-butoxycarbonyl-protected Leu-enkephalin resulted in a low yield of protected enkephalin amide.
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PMID:Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity. 1138 72

We developed a protocol for efficient expression of the functional serine protease, subtilisin E, in Escherichia coli periplasm that permits direct in vivo measurement of the enzyme's catalytic activity. Activity assays and SDS-PAGE/Western blot analysis showed that the levels of expressed subtilisin varied and were correlated with both the culture conditions and the induction procedures. The highest level of subtilisin expression was achieved at 0.10-0.15% (w/v) of arabinose as inducer and a temperature of 20-22 degrees C, and was ca. eightfold higher as compared to the expression level at 30 degrees C. Cultivation of bacterial cells to a steady state of balanced growth before induction was required for uniform subtilisin expression in cell cultures growing in wells of microtiter plates. Amidase and esterase cell-based kinetic assays on microtiter plates were developed based on the direct measurement of subtilisin activity in vivo. Intact E. coli cells displaying wild-type, dimethylformamide-resistant, and temperature-resistant subtilisins were assayed on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-acetyl-Phe-p-nitrophenyl ester for their amidase and esterase activity, respectively. Additionally, the periplasmic fractions were isolated from the three E. coli strains expressing the respective subtilisins and tested for amidase activity. The amidase activity of the three subtilisins was ca. 15-fold higher than the esterolytic activity when measured in both the intact cells and in the periplasmic fractions. The strategy combining periplasmic expression of subtilisins with two cell-based kinetic assays permits rapid screening of subtilisin mutant libraries for desired activities.
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PMID:A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm. 1200 Nov 68

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.
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PMID:Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods. 1662 Oct 88


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