Peptidase activity of β-lactamases

Although β-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of β-lactamases. The kcat/Km values were below 0.1 M-1˙s-1, but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best ‘peptidase’ was the class C β-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-D-Ala-D-Ala dipeptides than with the diacetyl- and α-acetyl-L-Lys-D-Ala-D-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble DD-peptidases. A comparison between the β-lactamases and DD-peptidases showed that it might be as difficult for a DD-peptidase to open the β-lactam ring as it is for the β-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

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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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ChemInform Abstract: FUNCTIONAL FLUOROCARBON MICELLES. SPECIFIC RATE ENHANCEMENT IN THE CATALYTIC HYDROLYSIS OF PHENYL ESTERS DUE TO SELECTIVE BINDING OF FLUOROCARBON AND HYDROCARBON SUBSTRATES

Chemischer Informationsdienst ◽

10.1002/chin.198421144 ◽

1984 ◽

Vol 15 (21) ◽

Author(s):

T. KUNITAKE ◽

H. IHARA ◽

Y. HASHIGUCHI

Keyword(s):

Specific Rate ◽

Catalytic Hydrolysis ◽

Selective Binding ◽

Rate Enhancement ◽

Hydrocarbon Substrates ◽

Hydrolysis Of

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Characterization of peptidases of adult Trichuris muris

Parasitology ◽

10.1017/s0031182000076502 ◽

1994 ◽

Vol 109 (5) ◽

pp. 623-630 ◽

Cited By ~ 16

Author(s):

L. J. Drake ◽

A. E. Bianco ◽

D. A. P. Bundy ◽

F. Ashall

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Matrix Protein ◽

Sodium Dodecyl ◽

Time Dependent ◽

Extracellular Matrix Protein ◽

Peptidase Activity ◽

Trichuris Muris ◽

Hydrolysis Of

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, Mr 85 and 105 kDa, which degrade gelatin optimally at pH 6·0 in sodium dodecyl sulphate–polyacrylamide gels. The peptidases were inactivated diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

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NB2001, a Novel Antibacterial Agent with Broad-Spectrum Activity and Enhanced Potency against β-Lactamase-Producing Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.5.1262-1268.2002 ◽

2002 ◽

Vol 46 (5) ◽

pp. 1262-1268 ◽

Cited By ~ 28

Author(s):

Qing Li ◽

Jean Y. Lee ◽

Rosario Castillo ◽

Mark S. Hixon ◽

Catherine Pujol ◽

...

Keyword(s):

Broad Spectrum ◽

Antibacterial Agent ◽

Bacterial Resistance ◽

Enterobacter Aerogenes ◽

Order Rate Constant ◽

Common Mechanism ◽

E Coli ◽

Lactam Ring ◽

Spectrum Activity ◽

Hydrolysis Of

ABSTRACT Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance resulting from enzyme overexpression. β-Lactamase overexpression is a common mechanism of bacterial resistance to β-lactam antibiotics. We present here the results for one of the β-lactamase ECTA compounds, NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold. Unlike conventional β-lactam antibiotics, where hydrolysis of the β-lactam ring inactivates the antibiotic, hydrolysis of NB2001 by β-lactamase releases triclosan. Evidence supporting the proposed mechanism is as follows. (i) NB2001 is a substrate for TEM-1 β-lactamase, forming triclosan with a second-order rate constant (k cat/Km ) of greater than 77,000 M−1 s−1. (ii) Triclosan is detected in NB2001-treated, β-lactamase-producing Escherichia coli but not in E. coli that does not express β-lactamase. (iii) NB2001 activity against β-lactamase-producing E. coli is decreased in the presence of the β-lactamase inhibitor clavulanic acid. NB2001 was similar to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum antibacterial agent and demonstrate the potential of ECTA in overcoming β-lactamase-mediated resistance.

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Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.543 ◽

1999 ◽

Vol 43 (3) ◽

pp. 543-548 ◽

Cited By ~ 13

Author(s):

Sonia Trépanier ◽

James R. Knox ◽

Natalie Clairoux ◽

François Sanschagrin ◽

Roger C. Levesque ◽

...

Keyword(s):

Three Dimensional ◽

Catalytic Efficiency ◽

Enterobacter Cloacae ◽

Acid Group ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Class C ◽

Three Dimensional Models ◽

Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

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Kinetic studies of modifier effects on the carboxypeptidase A catalyzed hydrolyses of peptides

Biochemistry and Cell Biology ◽

10.1139/o87-094 ◽

1987 ◽

Vol 65 (8) ◽

pp. 717-725 ◽

Cited By ~ 2

Author(s):

John F. Sebastian ◽

Richard S. Hinks ◽

Ralf V. Reuland

Keyword(s):

Kinetic Studies ◽

Competitive Inhibitor ◽

Peptidase Activity ◽

Benzene Sulfonate ◽

Carboxypeptidase A ◽

Structural Requirements ◽

Detailed Mechanism ◽

Hydrolysis Of ◽

Phenyl Alanine ◽

Standing Question

A variety of modifiers of carboxypeptidase A (CPA) have been investigated in an effort to understand the structural requirements of inhibitors and activators of peptidase activity. It is proposed that an understanding of the mechanism of action of reversible activators of the enzyme may bear on the long standing question of whether the detailed mechanism of peptidase activity is different from that of esterase activity. An analog of the activator 2,2-dimethyl-2-silapentane-5-sulfonate, 5,5-dimethylhexanoate, was found to be a competitive inhibitor of the CPA-catalyzed hydrolysis of benzoylglycyl-L-phenyl-alanine. The modifier 4-phenyl-3-butenoate (styrylacetic acid) was determined to be an activator. The sulfonates benzene-sulfonate, p-toluenesulfonate, phenylmethanesulfonate, 2-phenylethanesulfonate, and 3-phenylpropanesulfonate were all found to be activators.

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Hydrolysis of Human High-Molecular-Mass Kininogen by Human Plasma Kallikrein. Proposal of a New Model Concept for the Course of Reaction in Presence and Absence of C1-Inhibitor

Biological Chemistry Hoppe-Seyler ◽

10.1515/bchm3.1987.368.2.1203 ◽

1987 ◽

Vol 368 (2) ◽

pp. 1203-1214 ◽

Cited By ~ 2

Author(s):

Jürg WIEDLER ◽

Hans DUTLER

Keyword(s):

Molecular Mass ◽

Human Plasma ◽

High Molecular Mass ◽

Plasma Kallikrein ◽

C1 Inhibitor ◽

Model Concept ◽

New Model ◽

Presence And Absence ◽

Hydrolysis Of

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Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1966-1970.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1966-1970 ◽

Cited By ~ 32

Author(s):

Sergei B. Vakulenko ◽

Dasantila Golemi ◽

Bruce Geryk ◽

Maxim Suvorov ◽

James R. Knox ◽

...

Keyword(s):

Kinetic Parameters ◽

Broad Spectrum ◽

Random Mutagenesis ◽

Enterobacter Cloacae ◽

The Other ◽

Mutant Enzyme ◽

Wild Type ◽

Wild Type Enzyme ◽

Wide Range ◽

Class C

ABSTRACT The class C β-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat/Km ) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.

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