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A direct reading, six channel conductivity recording apparatus has been constructed and used to assay enzymes, including some of those found in muscle. This paper gives the essential theory of the method, and indicates that it is a highly versatile assay system. It may be applicable whenever ionic substrates are involved, but this is more easily tested by experiment than by calculation. The manipulation involved in an assay is probably less than in any other method. Conductimetry could be used as a kinetic method for some enzymes (for example amidases), but suffers from restrictions in the amounts of non-reacting ionic species which can be tolerated. Improvements in the temperature control of the apparatus could allow much higher sensitivity to be used. [ABSTRACT FROM AUTHOR]

The time-dependent inhibition of amidase from Pseudomonas aeruginosa strain AI 3 by urea, hydroxyurea and cyanate displayed saturation kinetics fitting a model for the reaction sequence in which formation of a complex in a reversible step was followed by an irreversible step. Altered amidases from mutant strains AIU 1N and OUCH 4, selected for their resistance to inhibition of growth by urea and hydroxyurea respectively, had altered kinetic constants for inhibition indicating reduced binding capacity for the inhibitors. The substrate acetamide protected AI 3 amidase against inhibition by urea, and altered Ki values for inhibition of the mutant amidases were paralleled by alterations in Km values for acetamide indicating that urea acted at the active site. Inhibition of AI 3 amidase involved the binding of one molecule of urea per molecule of enzyme. Urea inhibited amidase slowly regained activity at pH 7.2 through release of urea. [ABSTRACT FROM AUTHOR]

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant ( Lupinus luteus potassium-independent asparaginase, LlA) and bacterial ( Escherichia coli, ybiK/ spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (LlA) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg·L−1 of culture, respectively. The enzymes are heat-stable up to 60 °C and show both isoaspartyl dipeptidase and l-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue. [ABSTRACT FROM AUTHOR]

Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant ( kcat/ Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number ( kcat). [ABSTRACT FROM AUTHOR]

An N -acyl-d-amino acid amidohydrolase (N -D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N -acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxus N -D-AAase showed significant amino acid similarity to the N -acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44–56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N -D-AAase protein in Escherichia coli , the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U·mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N -D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 °C, respectively. After 30 min heat treatment at 45 °C, between pH 6 and pH 8, 80% activity remained. The N -D-AAase had higher hydrolysing activity against N -acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N -chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N -acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+ . Thus, the N -D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme. [ABSTRACT FROM AUTHOR]

Characterizes the D-stereospecific amino-acid amidase (DaaA) from Ochrobactrum anthropi SV3. Composition of enzyme, DaaA; Molecular mass of DaaA; Purification of the enzyme to electrophoretic homogeneity by ammonium sulfate fractionation; Similarity of DaaA characteristic with those found in native enzyme.

Characterizes an amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous. Sequencing and expression of the gene; Purification and characterization of the gene product.

Suggests that isopenicillin-N acyltransferase of Penicillin chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities. Encoding by the single penDE gene; Suggestion that the penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:g-APA acyltransferase.

5,6-Dihydropyrimidine amidohydrolase was isolated from an acetone powder of calf liver and purified to homogeneity. Purification made use of heat treatment, ammonium sulfate fractionation and chromatography on Chelating Sepharose and DEAE-Sepharose with 44% recovery of total activity. The native enzyme has a molecular mass of 217 kDa consisting of four subunits with a molecular mass of 54 kDa each. The amidohydrolase is a metalloenzyme containing one zinc atom/subunit. The enzyme can slowly be inactivated by chelating agents. The kinetic parameters for substrates, 5,6-dihydrouracil, 5,6-dihydrothymine and glutarimide were determined. From log Vmax/KM data, a pKa of 7.6 could be calculated suggesting the formation of a zinc-bound hydroxyl ion which carries out the nucleophilic attack on the C-4 of dihydrouracil. [ABSTRACT FROM AUTHOR]

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the β-lactam antibiotics. [ABSTRACT FROM AUTHOR]

Reports on the in vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes. Exhibition of amidase activity by the cell-free preparation; Binding to the oligosaccharide part of the lipopolysaccharide.

Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-γ-glutamyl-meso-diaminopimelic acid [MurNAc-LAla-DGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc-LAla linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1-2.5%) of cleaved MurNAc-LAla linkages were detected in peptidoglycan isolated from normally growing cells. However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful. Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E. coli as sole carbon and nitrogen sources. Finally, out of 200 thermosensitive mutants examined for altered amidase activity, only two strains had less than 50% of the normal level of activity, whereas ten strains were found to possess more than 50%. In fact, two of the overproducers encountered presented a 4--5-fold increase in activity. [ABSTRACT FROM AUTHOR]

N-Acetylmuramyl-L-alanine amidase activity was detected in Escherichia coli K12 by using N- acetylmuramyl-L-alanyl-γ-D-glutamyl-(L)-meso-[³H]diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N-acetylmuramyl acid and L-alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K12. The enzyme was purified to near homogeneity. A molecular weight of 39 000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N-acetylmuramyl-L-alanine amidase activity was carried out by investigating several of its properties. [ABSTRACT FROM AUTHOR]

Immunological resemblance between the endopeptidase, endo-β-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase of lysostaphin has been demonstrated. Only the peptidase was found to give precipitin reaction. All three enzymes sensitized tanned sheep erythrocytes to agglutination in homologous and heterologous enzyme antisera. Absorption experiments revealed the presence of specific determinant(s) in all the enzymes, determinant(s) shared between two and two enzymes, and a possible deterrninant(s)-common to all three enzymes. [ABSTRACT FROM AUTHOR]

The analysis of a monoclinic crystal modification of L-asparaginase from Eseherichia coli supports the chemical and physico-chemical evidence for a tetrameric subunit structure. The pseudo-symmetries occurring in the crystal structure provide evidence that the molecules lie on positions with approximate point symmetry 222. [ABSTRACT FROM AUTHOR]