The inhibition of staphylococcal β-lactamase by clavulanic acid

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.

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Inhibition of class C β-lactamases by (1′R,6R)-6-(1′-hydroxy)benzylpenicillanic acid SS-dioxide

Biochemical Journal ◽

10.1042/bj2250435 ◽

1985 ◽

Vol 225 (2) ◽

pp. 435-439 ◽

Cited By ~ 27

Author(s):

G C Knight ◽

S G Waley

Keyword(s):

Side Chain ◽

Beta Lactamase ◽

Beta Lactam ◽

Beta Lactamases ◽

Beta Lactam Antibiotics ◽

Lactam Ring ◽

Class C ◽

Hydrolysis Of

beta-Lactamases, enzymes that catalyse the hydrolysis of the beta-lactam ring in beta-lactam antibiotics, are divided into three classes, A, B and C, on the basis of the structures so far determined. There are relatively few effective inhibitors of class C beta-lactamases. A beta-lactam sulphone with a hydroxybenzyl side chain, namely (1′R,6R)-6-(1′-hydroxy)benzylpenicillanic acid SS-dioxide (I), has now been studied. The sulphone is a good mechanism-based inhibitor of class C beta-lactamases. At pH8, the inhibition of a Pseudomonas beta-lactamase is irreversible, and proceeds at a rate that is about one-tenth the rate of concurrent hydrolysis. The labelled enzyme has enhanced u.v. absorption and is probably an enamine. At a lower pH, however, inhibition is transitory.

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PREVALENCE OF CEPHALOSPORIN-RESISTANT GRAM-NEGATIVE BACILLI FROM CLINICAL SAMPLES

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2016.v9s2.13456 ◽

2016 ◽

pp. 176

Author(s):

Kavi Aniis ◽

Rajamanikandan Kcp ◽

Arvind Prasanth D

Keyword(s):

Clinical Samples ◽

Beta Lactamase ◽

Bacterial Isolates ◽

Beta Lactam ◽

Gram Negative ◽

Beta Lactamases ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Lactam Ring ◽

Esbl Producers

ABSTRACT Objective: Beta-lactams are the group of antibiotics that contain a ring called as “beta-lactam ring,” which is responsible for the antibacterial activity. The presence of resistance among Gram-negative organisms is due to the production of beta-lactamases enzymes that hydrolysis the beta-lactam ring thereby conferring resistance to the organism. This study is undertaken to determine the prevalence of extended-spectrum beta-lactamase (ESBL) producing Gram-negative organism from clinical samples. Methods: A total of 112 clinical samples were taken for this study. The combined disc synergistic test (CDST) was used for the phenotypic detection of ESBL producers from the clinical samples. The genotypic identification of ESBL producers was carried out by alkaline lysis method by isolation of plasmid DNA. Result: A total of 87 bacterial isolates were isolated and identified. Among them, Klebsiella (41%) was the predominant organism followed by Escherichia coli (33%), Proteus (10%), Pseudomonas (10%), and Serratia (6%). Among the various bacterial isolates, Klebsiella showed a higher percentage of resistance. The CDST showed that 8 isolates of Klebsiella, 3 isolates of E. coli, and 1 isolate of Pseudomonas were found to be ESBL producers. The genotypic confirmation showed that the two bacterial isolates, namely, Klebsiella and E. coli were found to possess temoniera (TEM) gene which was the 400-500 bp conferring resistance to the antibiotics. Conclusion: The results of this study suggest that early detection of ESBL producing Gram-negative organism is a very important step in planning the therapy of patient in Hospitals. CDST continues to be a good indicator in the detection of ESBL producers. Keywords: Beta-lactamases, Gram-negative bacilli, Extended-spectrum beta-lactamase, Resistance, Combined disc synergistic test.

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Characteristics of Aerobacter beta-lactamase

Canadian Journal of Microbiology ◽

10.1139/m68-023 ◽

1968 ◽

Vol 14 (2) ◽

pp. 139-145 ◽

Cited By ~ 4

Author(s):

M. Goldner ◽

D. G. Glass ◽

P. C. Fleming

Keyword(s):

Reaction Rates ◽

Penicillin G ◽

Cephalosporin C ◽

Beta Lactamase ◽

Beta Lactam ◽

Lactam Ring ◽

Rate Of Hydrolysis ◽

High Concentrations ◽

Hydrolysis Of ◽

Pseudomonas Pyocyanea

In this investigation, Aerobacter cloacae is shown to inactivate cephalosporin by hydrolysis of its beta-lactam ring. This was demonstrated by iodine absorption and infrared absorption spectra.The values of the Michaelis constant obtained with cephalosporin C and deacetyl cephalosporin C indicate a great affinity of the Aerobacter's beta-lactamase for its substrate. The enzyme was most active at pH 7.0 and 37 C. Aqueous washings of the Aerobacter cells were a potent source of enzyme.The beta-lactamase of A. cloacae was active on both cephalosporin and penicillin. A higher rate of hydrolysis was observed with cephalosporin C and deacetyl cephalosporin C than with cephalothin and cephaloridine. The ratio of reaction rates on cephalosporin C to that on penicillin G was consistently of the order of 100 to 1. The activity on V, N, and especially the semisynthetic penicillins was also low.The A. cloacae enzyme was easily demonstrable in large amount without added inducer. By contrast, the activity of the beta-lactamase from Pseudomonas pyocyanea cannot be detected unless high concentrations of inducer are used.

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Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

Biochemical Journal ◽

10.1042/bj2790111 ◽

1991 ◽

Vol 279 (1) ◽

pp. 111-114 ◽

Cited By ~ 3

Author(s):

J Martin Villacorta ◽

P Arriaga ◽

J Laynez ◽

M Menendez

Keyword(s):

Bacillus Cereus ◽

Kinetic Studies ◽

Beta Lactamase ◽

Beta Lactam ◽

Beta Lactamases ◽

Class A ◽

Rate Determining Step ◽

Class B ◽

Lactam Ring ◽

Beta Lactamase Ii

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

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Protein extracts from streptomyces sps. inhibits beta-lactamases secreted by pathogenic bacteria

International Journal of Advances in Pharmaceutical Sciences ◽

10.5138/09761055.2183 ◽

2018 ◽

Vol 9 (3) ◽

Author(s):

Rohit Mani Yadava ◽

Manjula Ishwara Kalyani

Keyword(s):

Enzyme Activity ◽

Pathogenic Bacteria ◽

Clinical Importance ◽

Beta Lactamase ◽

Bacterial Isolates ◽

Beta Lactam ◽

Biochemical Tests ◽

Beta Lactamases ◽

Culture Extract ◽

Dialysis Technique

Beta-lactamases are enzymes produced by pathogenic microorganisms which exhibit resistance to beta-lactam group of antibiotics and are of considerable clinical importance. In our study, we examined the pathogenic organisms for the secretion of beta-lactamase using the antibiotic ampicillin. The extracted beta-lactamase from the isolates was characterized biochemically for enzyme activity and to initiate their inhibition activities. The protein extracts separated from the potential actinomycetes species were analyzed by targeting against the beta lactamase enzyme activity. The beta-lactamase enzymes from bacterial isolates were purified from the cell free culture extract and activity was estimated spectrophotometrically. The actinomycetes isolated from the soil source were tested for their efficiency to inhibit the beta-lactamase enzyme activity. The protein fractions were extracted by salt precipitation using ammonium sulfate and further salt removal by dialysis technique. The assays for enzyme inhibition were performed by plate well diffusion along with absorbance readings of the enzyme and substrate using spectrophotometer. The beta-lactamase enzyme activity of Proteus sp. had shown highest enzymatic activity followed by Staphylococcus aureus and Pseudomonas sp. The protein extracts of four actinomycetes isolates that showed beta-lactamase inhibition were identified belonging to the genus Streptomyces based on their colony morphology, microscopic observation, and biochemical tests. The beta-lactamase inhibition activities were analyzed to combat antibiotitc resistances exerted by the pathogenic bacteria in infections.

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Interaction of clavulanate with the β-lactamases of Streptomyces albus G and Actinomadura R39

Biochemical Journal ◽

10.1042/bj2070429 ◽

1982 ◽

Vol 207 (3) ◽

pp. 429-436 ◽

Cited By ~ 33

Author(s):

J M Frère ◽

C Dormans ◽

V M Lenzini ◽

C Duyckaerts

Keyword(s):

Kinetic Models ◽

Reaction Scheme ◽

Beta Lactamase ◽

Beta Lactam ◽

General Reaction ◽

Beta Lactamases ◽

Complete Inactivation ◽

Streptomyces Albus ◽

Hydrolysis Of

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.

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The kinetics of non-stoichiometric bursts of β-lactam hydrolysis catalysed by class C β-lactamases

Biochemical Journal ◽

10.1042/bj2950295 ◽

1993 ◽

Vol 295 (1) ◽

pp. 295-304 ◽

Cited By ~ 22

Author(s):

M G P Page

Keyword(s):

Beta Lactam ◽

Beta Lactamases ◽

Beta Lactam Antibiotics ◽

Steady State Rate ◽

State Rate ◽

Rate Of Hydrolysis ◽

Class C ◽

Burst Kinetics ◽

Kinetics Of ◽

Hydrolysis Of

Class C beta-lactamases from Pseudomonas aeruginosa and several species of the Enterobacteriaceae have been observed to undergo a rapid burst in hydrolysis of beta-lactam antibiotics before relaxation to a steady-state rate of hydrolysis. The amplitude of the burst corresponds to the hydrolysis of between 1 and 10,000 mol of the substrate per mol of enzyme. The decay of the rate of hydrolysis in the burst phase comprises two exponential reactions, which indicates that there are three different reactive states of the enzymes. Examination of the kinetics of acylation by slowly reacting beta-lactams suggests that there are three forms of the free enzyme in slow equilibrium. Thus it would appear that the burst kinetics exhibited by class C enzymes can be attributed to redistribution of the enzyme between different conformations induced by the reaction with substrate.

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Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414

Biochemical Journal ◽

10.1042/bj2870583 ◽

1992 ◽

Vol 287 (2) ◽

pp. 583-588 ◽

Cited By ~ 16

Author(s):

J M Dominguez ◽

C Acebal ◽

J Jimenez ◽

I de la Mata ◽

R Macarron ◽

...

Keyword(s):

Enzyme Activity ◽

Trichoderma Reesei ◽

Time Course ◽

Enzyme Concentration ◽

Main Process ◽

Peptide Bonds ◽

First Order ◽

First Order Kinetics ◽

Endoglucanase I ◽

Hydrolysis Of

The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.

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Interaction of β-iodopenicillanate with the β-lactamases of Streptomyces albus G and Actinomadura R39

Biochemical Journal ◽

10.1042/bj2070437 ◽

1982 ◽

Vol 207 (3) ◽

pp. 437-444 ◽

Cited By ~ 20

Author(s):

J M Frère ◽

C Dormans ◽

C Duyckaerts ◽

J De Graeve

Keyword(s):

Bacillus Cereus ◽

Absorption Maximum ◽

Amide Bond ◽

Beta Lactamase ◽

Beta Lactam ◽

Beta Lactamases ◽

Streptomyces Albus ◽

Hydrolysis Of

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.

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Interactions between active-site-serine β-lactamases and compounds bearing a methoxy side chain on the α-face of the β-lactam ring: kinetic and molecular modelling studies

Biochemical Journal ◽

10.1042/bj2930607 ◽

1993 ◽

Vol 293 (3) ◽

pp. 607-611 ◽

Cited By ~ 32

Author(s):

A Matagne ◽

J Lamotte-Brasseur ◽

G Dive ◽

J R Knox ◽

J M Frère

Keyword(s):

Water Molecule ◽

Molecular Modelling ◽

Side Chain ◽

Beta Lactam ◽

Beta Lactamases ◽

Class A ◽

Molecular Modelling Studies ◽

Lactam Ring ◽

Modelling Studies

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When compared with the situation prevailing with good substrates, both acylation and deacylation steps appeared to be severely impaired. Molecular modelling studies of the structures of the Henri-Michaelis complexes and of the acyl-enzymes indicate a major displacement of the crystallographically observed water molecule which connects the glutamate-166 and serine-70 side chains and underline the role of this water molecule in both reaction steps.

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