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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.
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PMID:Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme. 0 71

Alkalimetric pH-stat titrations of cephalosporin C, cephacetril and their deacetyl derivatives using an acetyl esterase and beta-lactamase are described. The esterase was used to assay highly purified samples of cephalosporin C and cephacetril, and also to prepare analytically defined solutions of the deacetyl cephalosporins. Lactamase-catalyzed hydrolysis of the parent compounds was then found to generate exactly 2 equivalents of acid per mole; that of the deacetyl derivatives exactly 1 equivalent.
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PMID:Alkalimetric microassay of cephalosporins. 9 13

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.
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PMID:Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b. 77 Apr 29

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.
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PMID:Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase. 387 32

The penicillinase mediated by the R factor R1 in Escherichia coli has been purified and characterized. The purification procedure contained the following three steps: spheroplast formation, chromatography of the spheroplast supernatant fluid on DEAE cellulose, and preparative polyacrylamide-gel electrophoresis. The protein obtained gave only one band in analytical polyacrylamide-gel electrophoresis. To obtain milligram quantities of the enzyme, gel filtration on Sephadex G75 was run before the last step in the purification. By gel filtration on Sephadex G75, the molecular weight was estimated as 22,000. The pH optimum, tested in universal buffer, was 7.0. The turnover numbers for benzylpenicillin, d-ampicillin, and 6-aminopenicillanic acid were 4.2 x 10(4), 6.3 x 10(4), and 2.2 x 10(4) moles of substrate hydrolyzed per min by 1 mole of enzyme, whereas the Michaelis constants were 100, 160, and 440 mum, respectively. Cephalosporins were much poorer substrates for the R1 penicillinase than were the penicillins. The turnover number for cephalosporin C, cephaloridine, and 7-amino-cephalosporanic acid were 2.4 x 10(3), 5 x 10(2), and less than 2 x 10(2), respectively. These properties show that the R1 penicillinase is quite different from the chromosomally mediated penicillinase of E. coli (11). However, the R1 enzyme resembles another R-factor penicillinase previously purified by Richmond and Datta.
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PMID:Resistance of Escherichia coli to penicillins. VII. Purification and characterization of a penicillinase mediated by the R factor R1. 490 36

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.
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PMID:Cephalosporinase and penicillinase activities of a beta-lactamase from Pseudomonas pyocyanea. 586 14

Human liver alanine aminopeptidase (EC 3.4.11.14; L-alpha-aminoacyl-peptide hydrolase) catalyzes the stepwise hydrolysis of methionyl-lysyl-bradykinin to yield methionine, lysine, and the limit nonapeptide, bradykinin which is resistant to further hydrolytic cleavage by this enzyme. Alanine aminopeptidase also catalyzes the hydrolysis of various neutral amino acid beta-naphthylamides. This enzyme cleaves N-terminal arginyl residues unless the adjacent penultimate residue is proline as is the case for bradykinin. The properties are consistent with the requirements of a kinin converting enzyme. Human alanine aminopeptidase activity is reduced by several beta-lactam antibiotics, with the cloxacillin, oxacillin, and methicillin Ki values being 0.51 mM, 1.6 mM, and 2.4 mM respectively. Our experiments with radioactively labelled penicillin indicate that two moles of antibiotic are bound per mole of enzyme. Neither chromatography of the penicillin-treated enzyme on G-25 Sephadex, treatment of penicillin-G-treated enzyme with penicillinase, nor extensive dilution of cloxacillin-treated enzyme diminished the degree of inactivation produced. Inhibition was obtained with 6-aminopenicillanic acid, which indicated that the penicillin nucleus itself was being bound, but substitutions, as in cloxacillin, could enhance the binding.
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PMID:Human-liver alanine aminopeptidase. A kinin-converting enzyme sensitive to beta-lactam antibiotics. 612 21

Izumenolide is a potent inhibitor of beta-lactamases, especially from Gram-negative bacteria. The I50 value of 0.01 microgram/ml for TEM-2 beta-lactamase, after 10 min preincubation, corresponds to a ratio of 7.6 moles inhibitor per mole of enzyme. The initial inhibitory reaction with TEM-2 beta-lactamase exhibits mixed reaction kinetics, suggesting a possible overlapping binding site with the active center. Tem-2 beta-lactamase is irreversible inactivated by izumenolide in a biphasic reaction. Carbenicillin offers partial protection against inactivation. Izumenolide exhibits limited antibiotic activity against some Gram-negative bacteria. Against beta-lactamase producing bacteria izumenolide provides protection to ampicillin and cephaloridine but the protection is limited due to permeability problems associated with izumenolide entry into the cells.
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PMID:Izumenolide-a novel beta-lactamase inhibitor produced by Micromonospora. II. Biological properties. 697 39

In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 M-1.cm-1) and an intense feature at 320 nm (epsilon approximately 1570 M-1.cm-1). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.
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PMID:Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis. 881 Sep 19

A non-proline cis peptide is present between Glu166 and Ile167 in the active site of beta-lactamase from Staphylococcus aureus PC1. To examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166, the site-directed mutant N136A was produced. The enzyme shows no measurable hydrolytic activity toward a variety of penicillins or cephalosporins except for the chromogenic cephalosporin, nitrocefin. For nitrocefin, the progress curve exhibits a fast burst with a stoichiometry of 1 mol of degraded substrate per mole of enzyme followed by a slow phase with a hydrolysis rate that is reduced by approximately 700-fold compared with that of the wild-type enzyme. Thus, the mutant enzyme is deacylation defective. Monitoring the hydrolysis of nitrocefin after preincubation with a number of beta-lactam compounds shows that cephalosporins form stable acyl complexes with the enzyme, whereas penicillins do not. The molecular weight of the mutant was determined by electrospray mass spectrometry, and the presence of the stable acyl enzyme adducts with cephaloridine and cefotaxime was confirmed by both electrospray and MALDI mass spectrometry. Therefore, in addition to impairing deacylation, the acylation machinery has been altered compared with the wild-type enzyme to act on cephalosporins and not on penicillins. Urea denaturation and thermal unfolding studies show that the N136A mutant enzyme is less stable than the wild-type enzyme. However, stability against chemical denaturation of the mutant enzyme is enhanced in the presence of cephaloridine beyond the stability of the wild-type protein. This is attributed to accumulation of favorable interactions between the cephaloridine and the protein, which play a role in the folded state and not in the unfolded state.
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PMID:Probing the non-proline cis peptide bond in beta-lactamase from Staphylococcus aureus PC1 by the replacement Asn136 --> Ala. 928 75


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