Penicillin‐sensitive Enzymes and Penicillin‐binding Components in Bacterial Cells*

Strominger, Jack L.; Willoughby, Eileen; Kamiryo, Tatsuyuki; Blumberg, Peter M.; Yocum, R. Rogers

doi:10.1111/j.1749-6632.1974.tb43267.x

Cited by 48 publications

(27 citation statements)

References 15 publications

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“…They inhibit the formation of cross-links between adjacent peptide subunits of the polymer [2,3]. This final reaction in peptidoglycan synthesis is catalyzed by a transpeptidase which usually is considered to be the main killing site of these antibiotics [1,4,5]. …”

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confidence: 99%

Biosynthesis of Peptidoglycan in Gaffkya homari, The Mode of Action of Penicillin G and Mecillinam. The Mode of Action of Penicillin G and Mecillinam

Hammes

1976

Eur J Biochem

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The effect of the p-lactam antibiotics penicillin G and mecillinam on the incorporation of peptidoglycan into pre-formed cell wall peptidoglycan was studied with wall membrane enzyme preparations from GaJfya homari. Using UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) as precursors the incorporation of peptidoglycan into the pre-existing cell wall of G. homari was inhibited to an extent of 50% (ID50 value) at a concentration of 0.25 pg of penicillin G/ml. With UDP-GlcNAc and UDP-MurNAc-tetrapeptide as precursors the ID50 value was about 2500-fold greater (630 pg/ml). The inhibition by penicillin G of the incorporation of peptidoglycan from UDP-M~rNAc-[~~C]Lys-pentapeptide could be overcome by addition of non-radioactive UDP-MurNAc-tetrapeptide to the incubation mixture. In the presence of 5 pg of penicillin G/ml the incorporation of peptidoglycan formed from the mixture of [14C]alanine by transpeptidase activity. The enzyme preparation also exhibited DD-carboxypeptidase activity which was only slightly more sensitive to penicillin G and mecillinam than was the incorporation of peptidoglycan into the cell wall. Since the ID50 values for the p-lactam antibiotics are similar to the concentrations required to inhibit the growth of G. homari to an extent of 50 %, the DD-carboxypeptidase must be the killing site of both penicillin G and mecillinam /3-Lactam antibiotics are powerful inhibitors of the biosynthesis of peptidoglycan [l]. They inhibit the formation of cross-links between adjacent peptide subunits of the polymer [2,3]. This final reaction in peptidoglycan synthesis is catalyzed by a transpeptidase which usually is considered to be the main killing site of these antibiotics [1,4,5]. UDP-MU~NA~-AI~-DG~U-L~S-D['~C]A~~-D['~C]A~~ and non-radioactive UDP-MurNAc-tetrapeptide proceeded virtually without release of D-Other enzymes that are most sensitive to p-lactam antibiotics are DD-carboxypeptidases. They catalyze the hydrolysis of the ultimate D-alanine residue from peptides ending in a D-alanyl-D-alanine sequence [l] ; the function of this reaction is, however, only conjectural. Studies of the substrate requirements exhibited by the Dwcarboxypeptidase from Escherichia coli and phospho-MurNAc-pentapeptide are the best substrates for this enzyme when compared with other substrates bearing a pentapeptide moiety. The product formed from UDP-MurNAc-pentapeptide is UDP-MurNAc-tetrapeptide, a compound which has been isolated from various bacteria [7].As described in the preceding paper [8], UDPMurNAc-tetrapeptide is, surprisingly, more effectively utilized than UDP-MurNAc-pentapeptide for the formation of cell-wall-bound peptidoglycan by wall membrane enzyme preparations from Guffkya homari. It was further observed that the addition of UDP-MurNAc-tetrapeptide enhanced the incorporation of peptidoglycan formed from UDP-MurNAc-pentapeptide into cell wall. Therefore the presence of tetrapeptide subunits in the newly synthesized peptidoglycan should affect or even deter...

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Biosynthesis of Peptidoglycan in Gaffkya homari, The Mode of Action of Penicillin G and Mecillinam. The Mode of Action of Penicillin G and Mecillinam

Hammes

1976

Eur J Biochem

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“…In a brief report by Strominger el af. [12], it was claimed that the DD-carboxypeptidases from both Bacillus stearothermophilus and Bacillus subtilis hydrolysed benzylpenicillin to benzylpenicilloic acid and, hence, catalysed a reaction which was that of a penicillinase. Further experiments, however, did not confirm these preliminary findings and showed that benzylpenicilloic acid was not the reaction product (P. M. Blumberg, personal communication).…”

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confidence: 99%

Interaction between β‐Lactam Antibiotics and Exocellular dd‐Carboxypeptidase‐Transpeptidase of Streptomyces R61

Frère

Leyh‐Bouille

Ghuysen

et al. 1974

European Journal of Biochemistry

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On the basis of steady-state kinetics, inhibition of the exocellular Dmcarboxypeptidase-transpeptidase of Streptomyces R61 by /?-lactam antibiotics was competitive with regard to the donor substrate. However, the complexes formed between the Streptomyces R61 enzyme and various /I-lactam antibiotics were relatively stable, exhibiting half-lives of 40 to 80 min at 37 "C and neutral pH. During breakdown of the complexes the protein underwent reactivation, whereas the released antibiotic molecule was chemically altered. With ['4C]benzylpenicillin, the released compound was neither benzylpenicillin nor benzylpenicilloic acid. The properties of the Streptomyces R61 enzyme . B-lactam antibiotic complexes were compared with those of the complexes formed between the same antibiotics and either the membrane-bound transpeptidase from Streptomyces R61 or the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39.Streptomyces strain R61 excretes during growth an exocellular DD-carboxypeptidase-transpeptidase which is thought to be a soluble form of the membranebound transpeptidase involved in the biosynthesis of the wall peptidoglycan [l]. Kinetically, benzylpenicillin was found to inhibit the DD-carboxypeptidase activity of this exocellular enzyme in a competitive manner with a K i value of 80nM [2]. Fluorimetric studies also suggested that the interaction between the Streptomvces R61 enzyme and benzylpenicillin was of the type E + P + E P where kf (the second-order constant for the forward reaction) had a value of 104 1 x mol-' x s-' and k , (the first-order constant for the reverse reaction) a value of x SC' [3]. The Ki value (k,/k,) thus obtained (at 25 "C) was equal to 10 nM and was in fair agreement with the above Ki value obtained by steady-state kinetics (at 37 "C). However, the value of the constant for the reverse reaction was much too small to account for a rapid equilibrium of the reaction system and suggested that the complex formed between the Streptomyces R61 enzyme and the antibiotic had a MATERIALS AND METHODS EnzymesThe Streptomyces R61 DD-carboxypeptidase-transpeptidase had a specific activity of 86 units/mg protein as determined in the carboxypeptidase assay (Ac2- L

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“…Native penicilloyl-carboxypeptidase is required for the release reactions, suggesting that these reactions are enzymatically catalyzed (8). The penicillin derivatives released in the presence of hydroxylamine or ethanethiol have been identified (6), whereas the spontaneously released product was not identified (7,8 Fig. 3, mass detector recording) showed a single peak with an equivalent chain length of C-14.4 (SE-30 column).…”

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“…Native penicilloyl-carboxypeptidase is required for the release reactions, suggesting that these reactions are enzymatically catalyzed (8). The penicillin derivatives released in the presence of hydroxylamine or ethanethiol have been identified (6), whereas the spontaneously released product was not identified (7,8). This report describes the identification of the spontaneously released [8-14C]penicillin G degradation product as phenylacetylglycine based on thin-layer and gasliquid chromatography, infrared spectroscopy, and mass spectrometry.…”

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Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.

Hammarström¹,

Strominger²

1975

Proc. Natl. Acad. Sci. U.S.A.

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(3,4), an enzyme which catalyzes a reaction analogous to the transpeptidation. In this reaction water rather than the amino-group of a pentaglycine chain serves as acceptor for the acyl-D-alanyl residue. D-Alanine carboxypeptidase is also inhibited by penicillins which bind covalently to the enzyme (4, 5). This binding may be reversed by hydroxylamine or ethanethiol (6) or spontaneously (7,8). Native penicilloyl-carboxypeptidase is required for the release reactions, suggesting that these reactions are enzymatically catalyzed (8). The penicillin derivatives released in the presence of hydroxylamine or ethanethiol have been identified (6), whereas the spontaneously released product was not identified (7,8 Fig. 3, mass detector recording) showed a single peak with an equivalent chain length of C-14.4 (SE-30 column). The mass spectrum (Fig. 5, Product. B. stearothermophilus, strain ATCC 15952, was grown as previously described (4). Membranes were prepared by grinding the cells with glass beads and differential centrifugation (3). The membranes were resuspended in 0.05 M Tris-HCl buffer, pH 7.5, to a protein concentration of 20 mg/ml. Five hundred milliliters of this suspension and 1 ml cephalothin (1 mg/ml) were incubated at 370 for 10 min. After this, 1 ml of [8-14C]penicillin G (1.5 mg = 4.2 ,gmol; specific activity: 3.8 mCi/mmol) was added and incubation was continued for another 10 min. The mixture was then centrifuged at 100,000 X g for 1 hr, the pellet was washed once with 0.05 M Tris, pH 7.5, resuspended in 200 ml of 0.01 M sodium cacodylate buffer, pH 6.5, and incubated at 550 for 50 min. After addition of 200 ml of water, the suspension was centrifuged at 100,000 X g for 1 hr. The pellet was discarded and the supernatant solution was Iyophilized. The residue thus obtained was dissolved in 0.02 N HCI and extracted twice with ethyl acetate. The combined extracts were concentrated to dryness in vacuo and the residue was stored at -20°(yield: 0.67 gCi-0. 18 ,imol). Part of the release product was treated with diazomethane and purified by preparative thin-layer chromatography (see below). Stability of Benzylpenicilloic Acid at pH 6.5 and 55°. Abbreviations: m.p., melting point; m/e, mass to charge ratio.

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Penicillin‐sensitive Enzymes and Penicillin‐binding Components in Bacterial Cells*

Cited by 48 publications

References 15 publications

Biosynthesis of Peptidoglycan in Gaffkya homari, The Mode of Action of Penicillin G and Mecillinam. The Mode of Action of Penicillin G and Mecillinam

Biosynthesis of Peptidoglycan in Gaffkya homari, The Mode of Action of Penicillin G and Mecillinam. The Mode of Action of Penicillin G and Mecillinam

Interaction between β‐Lactam Antibiotics and Exocellular dd‐Carboxypeptidase‐Transpeptidase of Streptomyces R61

Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.

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