scholarly journals Inactivation of TEM-1 β-lactamase by 6-acetylmethylenepenicillanic acid

1983 ◽  
Vol 209 (3) ◽  
pp. 609-615 ◽  
Author(s):  
M Arisawa ◽  
R Then
Keyword(s):  
Rate Constant ◽  
Active Site ◽  
Native Enzyme ◽  
Stable Form ◽  
Beta Lactamase ◽  
Irreversible Inhibitor ◽  
Beta Lactamases ◽  
Inactivation Constant ◽  
Inactivation Process ◽  
Fold Excess

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

scholarly journals A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

scholarly journals Phosphonate monoester inhibitors of class A β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 793-795 ◽  
Author(s):  
J Rahil ◽  
R F Pratt
Keyword(s):  
Enzyme Inhibition ◽  
Mechanism Of Action ◽  
Active Site ◽  
General Structure ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Inhibitory Ability ◽  
Class A ◽  
Structure Formula ◽  
Slow Turnover

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

Enzymatic activity is necessary for thrombin-mediated increase in endothelial permeability

1990 ◽  
Vol 259 (4) ◽  
pp. L270-L275 ◽  
Author(s):  
J. L. Aschner ◽  
J. M. Lennon ◽  
J. W. Fenton ◽  
M. Aschner ◽  
A. B. Malik
Keyword(s):  
Active Site ◽  
Endothelial Permeability ◽  
Covalent Bond ◽  
Catalytic Site ◽  
Irreversible Inhibitor ◽  
Chloromethyl Ketone ◽  
High Concentrations ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Fold Excess

alpha-Thrombin causes a dose-dependent increase in endothelial permeability as measured by the clearance rate of 125I-albumin across a monolayer of bovine pulmonary artery endothelial cells. We determined if an active catalytic site is necessary for the thrombin-mediated increase in endothelial permeability. alpha-Thrombin was reacted with 10-fold excess D-phenylalanyl-prolyl-arginine chloromethyl ketone (PPACK), an irreversible inhibitor that forms a covalent bond with thrombin's active site, producing an enzymatically inactive thrombin. PPACK completely inhibited the alpha-thrombin-mediated increase in 125I-albumin permeability. Similar results were obtained with gamma-thrombin, an enzymatically active alpha-thrombin form with an altered fibrinogen recognition domain. PPACK alone and the active site-inhibited PPACK-alpha-thrombin had no effect on permeability. Diisopropylphospho (DIP)-alpha-thrombin was effective only in very high concentrations (10(-6)M), and this effect was abolished by the addition of PPACK. These studies demonstrate that binding alone is insufficient for the thrombin-mediated increase in endothelial monolayer permeability. Thrombin's active catalytic site is a requirement for the increase in transendothelial albumin permeability.

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 The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals Active sites of β-lactamases. The chromosomal β-lactamases of Pseudomonas aeruginosa and Escherichia coli

1982 ◽  
Vol 201 (3) ◽  
pp. 621-627 ◽  
Author(s):  
V Knott-Hunziker ◽  
S Petursson ◽  
G S Jayatilake ◽  
S G Waley ◽  
B Jaurin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Active Sites ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
The Press ◽  
K 12

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.

scholarly journals KPC Beta-Lactamases Are Permissive to Insertions and Deletions Conferring Substrate Spectrum Modifications and Resistance to Ceftazidime-Avibactam

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Claire Amaris Hobson ◽  
Stéphane Bonacorsi ◽  
Hervé Jacquier ◽  
Alaksh Choudhury ◽  
Mélanie Magnan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Beta Lactamase ◽  
Content Type ◽  
Beta Lactamases ◽  
Extended Spectrum Beta Lactamase ◽  
Insertions And Deletions ◽  
Resistant Mutants ◽  
Substrate Spectrum

ABSTRACT To explore the mutational possibilities of insertions and deletions (indels) in the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase, we selected for ceftazidime-avibactam-resistant mutants. Of 96 screened mutants, we obtained 19 indels (2 to 15 amino acids), all located in the loops surrounding the active site. Three antibiotic susceptibility phenotypes emerged: an extended-spectrum-beta-lactamase-like phenotype, an activity restricted to ceftazidime, and a carbapenem-susceptible KPC-like phenotype. Tolerance for indels reflects the evolvability of KPC beta-lactamase, which could challenge the therapeutic management of patients.

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 β-lactamase inhibitors. The inhibition of serine β-lactamases by specific boronic acids

1988 ◽  
Vol 251 (2) ◽  
pp. 453-459 ◽  
Author(s):  
I E Crompton ◽  
B K Cuthbert ◽  
G Lowe ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Boronic Acids ◽  
Penicillin G ◽  
Side Chain ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Step Mechanism

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.

scholarly journals Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of β-lactamase

1992 ◽  
Vol 281 (1) ◽  
pp. 191-196 ◽  
Author(s):  
A K Tan ◽  
A L Fink
Keyword(s):  
Staphylococcus Aureus ◽  
Rate Constant ◽  
Active Site ◽  
Covalent Attachment ◽  
Beta Lactamase ◽  
Peptide Fragment ◽  
Serine Residue ◽  
Complete Inactivation ◽  
Enzyme Intermediate ◽  
Inhibited Enzyme

Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates.

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