scholarly journals Inactivation of the thiol RTEM-1 β-lactamase by 6-β-bromopenicillanic acid. Identity of the primary active-site nucleophile

1987 ◽  
Vol 247 (1) ◽  
pp. 29-33 ◽  
Author(s):  
A K Knap ◽  
R F Pratt
Keyword(s):  
Clavulanic Acid ◽  
Active Site ◽  
Thiol Group ◽  
Native Enzyme ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Thiol Ester ◽  
Acid Identity ◽  
Model Studies ◽  
Enzyme Model

The thiol RTEM-1 beta-lactamase [Sigal, Harwood & Arentzen (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme. Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate. Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group. These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover. The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm. Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently. Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase.

scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

scholarly journals Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

1997 ◽  
Vol 41 (7) ◽  
pp. 1460-1464 ◽  
Author(s):  
T R Walsh ◽  
A P MacGowan ◽  
P M Bennett
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Leader Peptide ◽  
Beta Lactamase ◽  
Class A ◽  
Cloned Gene ◽  
Kinetics Of

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

scholarly journals Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 β-lactamase II

1991 ◽  
Vol 276 (2) ◽  
pp. 401-404 ◽  
Author(s):  
H M Lim ◽  
R K Iyer ◽  
J J Pène
Keyword(s):  
Bacillus Cereus ◽  
Aspartic Acid ◽  
Carboxy Group ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Aspartic Acid Residue ◽  
Beta Lactamase Ii

An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an ‘in vivo’ study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

scholarly journals Assessment of two penicillins plus beta-lactamase inhibitors versus cefotaxime in treatment of murine Klebsiella pneumoniae infections.

1996 ◽  
Vol 40 (2) ◽  
pp. 325-330 ◽  
Author(s):  
J L Fournier ◽  
F Ramisse ◽  
A C Jacolot ◽  
M Szatanik ◽  
O J Petitjean ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clavulanic Acid ◽  
Survival Rates ◽  
Dose Effect ◽  
Pharmacokinetic Analysis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Bacterial Killing ◽  
Bacterial Counts

The in vivo efficacies of piperacillin, piperacillin plus tazobactam, ticarcillin, ticarcillin plus clavulanic acid, piperacillin plus clavulanic acid, and cefotaxime were compared in a mouse model of pneumonia induced by the SHV-1 beta-lactamase-producer Klebsiella pneumoniae. Each antibiotic was injected either once intraperitoneally at 24 h postinfection or at repeated times during 24 h. The efficacies of the drugs and therapeutic protocols were assessed by counting viable bacteria recovered from the lungs of mice sacrificed at selected times. No emergence of beta-lactam-resistant organisms was detected. Ticarcillin at 300 mg/kg was ineffective. Repeated injections of piperacillin at 300 mg/kg, either alone or in combination with tazobactam (8:1), led to a significant decrease in bacterial counts, but this was followed by bacterial regrowth. The pharmacokinetic analysis demonstrated that this short-lasting antibacterial effect was not due to a failure of piperacillin and/or tazobactam to penetrate the lungs. The combinations of ticarcillin at 300 mg/kg plus clavulanic acid (15:1) and piperacillin at 300 mg/kg plus tazobactam (4:1) were proven to be effective in that they decreased the bacterial burden in the lungs from 10(5) to < 10(3) CFU. This dose effect of tazobactam can be explained by its dose-dependent penetration in the lungs. Cefotaxime at 100 mg/kg and the combination of piperacillin (slightly hydrolyzed by SHV-1) at 300 mg/kg plus clavulanic acid (15:1) led to the best efficacy. Both of these treatments induced a decrease in bacterial counts of nearly 4 log10 units. The survival rates correlated with the quantitative measurements of in vivo bacterial killing. These experimental results obtained from the restricted animal model used here may help in the design of further protocols for clinical trials.

scholarly journals A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate

1989 ◽  
Vol 258 (3) ◽  
pp. 765-768 ◽  
Author(s):  
B P Murphy ◽  
R F Pratt
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Direct Interaction ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Enzyme Active Site ◽  
Lactam Carbonyl ◽  
Beta Lactamase Ii ◽  
Hydrolysis Of

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.

scholarly journals Interactions between active-site-serine β-lactamases and mechanism-based inactivators: a kinetic study and an overview

1993 ◽  
Vol 295 (3) ◽  
pp. 705-711 ◽  
Author(s):  
A Matagne ◽  
M F Ghuysen ◽  
J M Frère
Keyword(s):  
Kinetic Study ◽  
Clavulanic Acid ◽  
Active Site ◽  
Rate Constants ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.

scholarly journals Kinetics of inactivation of β-lactamase I by 6 β-bromopenicillanic acid

1980 ◽  
Vol 187 (3) ◽  
pp. 797-802 ◽  
Author(s):  
V Knott-Hunziker ◽  
B S Orlek ◽  
P G Sammes ◽  
S G Waley
Keyword(s):  
Bacillus Cereus ◽  
Rate Constant ◽  
Active Site ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Rate Determining Step ◽  
Competitive Model ◽  
Lactam Ring ◽  
Acid Inactivation ◽  
Kinetics Of

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.

scholarly journals Beta-lactamase inhibitors from laboratory to clinic.

1988 ◽  
Vol 1 (1) ◽  
pp. 109-123 ◽  
Author(s):  
K Bush
Keyword(s):  
Side Effects ◽  
Clavulanic Acid ◽  
Broad Spectrum ◽  
Pathogenic Bacteria ◽  
Defense Mechanism ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Beta Lactam Antibiotics ◽  
Tract Infections

beta-Lactamases constitute the major defense mechanism of pathogenic bacteria against beta-lactam antibiotics. When the beta-lactam ring of this antibiotic class is hydrolyzed, antimicrobial activity is destroyed. Although beta-lactamases have been identified with clinical failures for over 40 years, enzymes with various abilities to hydrolyze specific penicillins or cephalosporins are appearing more frequently in clinical isolates. One approach to counteracting this resistance mechanism has been through the development of beta-lactamase inactivators. beta-Lactamase inhibitors include clavulanic acid and sulbactam, molecules with minimal antibiotic activity. However, when combined with safe and efficacious penicillins or cephalosporins, these inhibitors can serve to protect the familiar beta-lactam antibiotics from hydrolysis by penicillinases or broad-spectrum beta-lactamases. Both of these molecules eventually inactivate the target enzymes permanently. Although clavulanic acid exhibits more potent inhibitory activity than sulbactam, especially against the TEM-type broad-spectrum beta-lactamases, the spectrum of inhibitory activities are very similar. Neither of these inhibitors acts as a good inhibitor of the cephalosporinases. Clavulanic acid has been most frequently combined with amoxicillin in the orally active Augmentin and with ticarcillin in the parenteral beta-lactam combination Timentin. Sulbactam has been used primarily to protect ampicillin from enzymatic hydrolysis. Sulbactam has been used either in the orally absorbed prodrug form as sultamicillin or as the injectable combination ampicillin-sulbactam. Synergy has been demonstrated for these combinations for most members of the Enterobacteriaceae, although those organisms that produce cephalosporinases are not well inhibited. Synergy has also been observed for Neisseria gonorrhoeae, Haemophilus influenzae, penicillinase-producing Staphylococcus aureus, and anaerobic organisms. These antibiotic combinations have been used clinically to treat urinary tract infections, bone and soft-tissue infections, gonorrhea, respiratory infections, and otitis media. Gastrointestinal side effects have been reported for Augmentin and sultamicillin; most side effects with these agents have been mild. Although combination therapy with beta-lactamase inactivators has been used successfully, the problem of resistance development to two agents must be considered. Induction of cephalosporinases can occur with clavulanic acid. Permeability mutants could arise, especially with added pressure from a second beta-lactam.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli to 12 beta-lactam agents and combinations with beta-lactamase inhibitors.

1996 ◽  
Vol 40 (8) ◽  
pp. 1924-1925 ◽  
Author(s):  
P Tajada ◽  
J L Gomez-Graces ◽  
J I Alós ◽  
D Balas ◽  
R Cogollos
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Campylobacter Coli ◽  
Beta Lactamase ◽  
In Vitro Activity ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Thermophilic Campylobacter ◽  
Amoxicillin Clavulanic Acid

The in vitro activities of 12 beta-lactam agents against 100 thermophilic Campylobacter strains were tested. Beta-Lactamase production was detected in 88% of all strains tested. Clavulanic acid was the best inhibitor by susceptibility testing. The beta-lactams which displayed high levels of in vitro activity against Campylobacter isolates were imipenem, amoxicillin-clavulanic acid, and cefepime and, to a lesser degree, amoxicillin, ampicillin, and cefotaxime.

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