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

1993 ◽  
Vol 295 (1) ◽  
pp. 295-304 ◽  
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.

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

1985 ◽  
Vol 225 (2) ◽  
pp. 435-439 ◽  
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.

scholarly journals Substrate-induced inactivation of the OXA2 β-lactamase

1993 ◽  
Vol 295 (3) ◽  
pp. 871-878 ◽  
Author(s):  
P Ledent ◽  
J M Frère
Keyword(s):  
Competitive Inhibition ◽  
Hydrolysis Product ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Hydrolysis Time ◽  
Beta Lactam Antibiotics ◽  
Class D ◽  
Time Courses ◽  
Burst Kinetics ◽  
Hydrolysis Of

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. ‘Burst’ kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two ‘bursts’, and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.

A New Method for Determining Individual Rate Constants for Specific Non-chromophoric Substrates of Proteolytic Enzymes

1975 ◽  
Vol 53 (4) ◽  
pp. 564-571
Author(s):  
Lewis J. Brubacher
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Proteolytic Enzymes ◽  
Enzyme Mechanism ◽  
Steady State Kinetics ◽  
Steady State Rate ◽  
State Rate ◽  
Individual Rate ◽  
Kinetics Of ◽  
Hydrolysis Of

Equations are developed for the pre-steady state kinetics of the proteolytic enzyme-catalyzed hydrolysis of a substrate A in the presence of a monitoring substrate (or covalent inhibitor) S of known properties. A two-intermediate acyl–enzyme mechanism is assumed in which the first intermediate is in instantaneous equilibrium with enzyme and substrate. The appearance of the first product of substrate S is characterized by two relaxation rate constants. From these constants it is possible to determine the dissociation constant and the acylation and deacylation rate constants of substrate A. Criteria are also developed for using the steady state rate parameters of A to establish conditions for which the slower relaxation process is equivalent to the deacylation rate constant of A. The technique of premixing enzyme with substrate A has certain advantages in this approach.

scholarly journals Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase

1993 ◽  
Vol 90 (23) ◽  
pp. 11257-11261 ◽  
Author(s):  
E Lobkovsky ◽  
P C Moews ◽  
H Liu ◽  
H Zhao ◽  
J M Frere ◽  
...  
Keyword(s):  
Binding Site ◽  
Enterobacter Cloacae ◽  
Crystallographic Structure ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
X Ray Crystallography ◽  
Class A ◽  
Beta Lactam Antibiotics ◽  
Class C

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

scholarly journals Phenotypic detection of Carbapenamase among Klebsiella pneumoniae isolated from clinical samples using modified Hodged test

2020 ◽  
Vol 13 (3) ◽  
pp. 135-140
Author(s):  
HauwaYakubu ◽  
Mahmud Yerima Iliyasu ◽  
Asma’u Salisu ◽  
Abdulmumin Ibrahim Sulaiman ◽  
Fatima Tahir ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Susceptibility Testing ◽  
University Teaching ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Beta Lactam Antibiotics ◽  
Microbiological Techniques ◽  
Tract Infections

Carbapenemases are microbial enzymes that confer resistance to virtually all available beta-lactam antibiotics and the most frequent carbapenemases are the Klebsiella pneumoniae Carbapenamase (KPC). Detection of carbapenemases is a significant infection control strategy as the enzymes are often associated with extensive antimicrobial resistance, therapeutic failures and mortality associated with infectious diseases. A total of 400 clinical samples were collected from different groups of patients in Abubakar Tafawa Balewa University Teaching Hospital, Bauchi, Nigeria and 118 K. pneumoniae were isolated using standard microbiological techniques. The isolates were subjected to antibiotic susceptibility testing by Kirby-Bauer disc diffusion method, then screened for Carbapenamase production using modified Hodge test. The results indicated that the isolates were resistant to Ampicillin (61.9%), Ceftriaxone (50.8%) and Ceftazidime (50.8%), then Ciprofloxacin (54.2%), but predominantly sensitive to Imipenem (66.9%), Eterpenem (60.2%) and Meropenem (65.3%). It was found that 38 (32.2%) of the isolates phenotypically shows the presence of Carbapenamase, with highest frequency of (40.7%) among patients, mainly adult females with cases of Urinary Tract Infections (UTIs) and the least from wound (11.8%).This study revealed that the isolates produced other beta-lactamases than KPC or variants of Carbapenamase that cannot be detected by modified Hodge test, thus shows low resistance to carbapenems. Therefore further studies is needed to genotypically confirm the presence of KPC in these isolates.

scholarly journals A metallo-beta-lactamase with both beta-lactamase and ribonuclease activity is linked with traduction in giant viruses

2019 ◽  
Author(s):  
Philippe Colson ◽  
Lucile Pinault ◽  
Said Azza ◽  
Nicholas Armstrong ◽  
Eric Chabriere ◽  
...  
Keyword(s):  
Acanthamoeba Castellanii ◽  
Deep Ocean ◽  
Penicillin G ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Strong Activity ◽  
Beta Lactam Antibiotics ◽  
Double Stranded Dna ◽  
Giant Viruses ◽  
Hydrolysis Of

ABSTRACTEnzymatic proteins with a metallo-beta-lactamase (MBL) fold have been essentially studied in bacteria for their activity on beta-lactam antibiotics. However, the MBL fold is ancient and highly conserved, and these proteins are capable of cleaving a broad range of substrates. It has recently been shown that MBLs are present in a wide array of cellular organisms, including eukaryotes and archaea. We show here that Tupanvirus deep ocean, a giant virus, also encodes a protein with a MBL fold. Phylogeny showed its clustering with transfer ribonucleases (RNases) and the presence of orthologs in other giant viruses, mainly those harboring the largest sets of translation components. In addition, it suggests an ancient origin for these genes and a transfer between giant viruses and Acanthamoeba spp., a host of many giant viruses. Biologically, after its expression in Escherichia coli, the tupanvirus protein was found to hydrolyse nitrocefin, a chromogenic beta-lactam. We also observed an hydrolysis of penicillin G (10 μg/mL) and detected the metabolite of penicillin G hydrolysis, benzylpenilloic acid. This was inhibited by sulbactam, a beta-lactamase inhibitor. In addition, we tested the degradation of single-stranded DNA, double-stranded DNA, and RNAs, and observed a strong activity on RNAs from seven bacteria with G+C varying from 42% to 67%, and from Acanthamoeba castellanii, the tupanvirus host. This was not inhibited by sulbactam or ceftriaxone. RNase activity was estimated to be 0.45±0.15 mU/mg using a fluorescence-based assay. Our results still broaden the range of hosts of MBL fold proteins and demonstrate that such protein can have dual beta-lactamase/nuclease activities. We suggest that they should be annotated according to this finding to avoid further confusion.

scholarly journals Beta-lactamase of Mycobacterium fortuitum: kinetics of production and relationship with resistance to beta-lactam antibiotics.

1991 ◽  
Vol 35 (9) ◽  
pp. 1760-1764 ◽  
Author(s):  
L Fattorini ◽  
G Scardaci ◽  
S H Jin ◽  
G Amicosante ◽  
N Franceschini ◽  
...  
Keyword(s):  
Mycobacterium Fortuitum ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactam Antibiotics ◽  
Kinetics Of

scholarly journals Imipenem as substrate and inhibitor of β-lactamases

1988 ◽  
Vol 253 (2) ◽  
pp. 323-328 ◽  
Author(s):  
J Monks ◽  
S G Waley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacillus Cereus ◽  
Reversible Reaction ◽  
Clinical Use ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Class C ◽  
Kinetics Of ◽  
Carbapenem Antibiotic

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.

Mechanistic Information from the Effect of Pressure on the Kinetics of Hydrolysis of Iodopentaaquochromium(III) Ion

1975 ◽  
Vol 53 (24) ◽  
pp. 3697-3701 ◽  
Author(s):  
Milton Cornelius Weekes ◽  
Thomas Wilson Swaddle
Keyword(s):  
Ionic Strength ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Hydrogen Ion Concentration ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Aqueous Hclo ◽  
Conjugate Base

The rate of hydrolysis of iodopentaaquochromium(III) ion has been measured as a function of pressure (0.1 to 250 MPa) and hydrogen ion concentration (0.1 to 1.0 mol kg−1) at 298.2 K and ionic strength 1.0 mol kg−1 (aqueous HClO4–LiClO4). The volumes of activation for the acid independent and inversely acid dependent hydrolysis pathways are −5.4 ± 0.5 and −1.6 ± 0.3 cm3 mol−1 respectively, and are not detectably pressure-dependent. Consideration of these values, together with the molar volume change of −3.3 ± 0.3 cm3 mol−1 determined dilatometrically for the completed hydrolysis reaction, indicates that the mechanisms of the two pathways are associative interchange (Ia) and dissociative conjugate base (Dcb) respectively.

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