Beam Loss Due to G10 Targeting

Blumberg, L.; Glenn, J.W.; Hornstra, F.; Keane, J.; Lee, Y.Y.; Month, M.; Smith, L.; Soukas, A.

doi:10.2172/1151035

Cited by 3 publications

(6 citation statements)

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“…antibiotic complexes were described [7-101. Contrary to a suggestion made by Blumberg and Strominger [7], the present studies support the idea that all DD-carboxypeptidases and transpeptidases are probably inhibited by penicillin according to the same general mechanism, although, depending upon the enzyme andjor the technique used, the inhibition inay appear as 'competitive' (in Lineweaver-Burk plots) or as 'irreversible' ( i .~. involving the forination of rather stable complexes EI").…”

Section: Discussioncontrasting

confidence: 89%

“…1 shows plots obtained with carbenicillin and penicillin V. Moreover, the Dixon plots of both theoretical and experimental data yielded converging lines from which the following apparent Ki values were calculated : carbenicillin 0.73 pM (theoretical) and 1.05 pM (experimental); penicillin V 0.33 pM (theoretical and experimental) ; benzylpenicillin 61.5 nM (theoretical) and 80 nM (experimental). It should be noted that these apparent K , values were significantly higher than those which would have been obtained from only the steady-state term of Eqn (7). Such apparent K , values [K, = k,K/ (k3 + k4)] were 0.17pM, 93 nM and 10nM for carbenicillin, penicillin V and benzylpenicillin respectively.…”

Section: With Each Of These Antibiotics the Conditions [El @ [I] Andmentioning

confidence: 68%

“…Such apparent K , values [K, = k,K/ (k3 + k4)] were 0.17pM, 93 nM and 10nM for carbenicillin, penicillin V and benzylpenicillin respectively. As shown in Table 2, these differences were due to the fact that the two non-steady-state terms of Eqn (7) were not negligible with regard to the steadystate term. In the case of benzylpenicillin the contribution of the two non-steady-state terms were 3 to 4 times more important than that of the steady-state term.…”

Section: With Each Of These Antibiotics the Conditions [El @ [I] Andmentioning

confidence: 99%

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Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics

Frère

Ghuysen

Perkins

1975

European Journal of Biochemistry

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The interaction between the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61 and p-lactam antibiotics is a multistep process during which a rather stable enzyme . antibiotic complex is formed. This mechanism of interaction is compatible with Lineweaver-Burk plots that are typical of a competitive inhibition of the hydrolysis of the peptide donor by the antibiotic. In fact, however, the same Lineweaver-Burk plots can be obtained on the basis of a non-competitive type of inhibition. At present, a choice between the two models cannot be made.Recent studies showed that the interaction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomvces R61 (E) and p-lactam antibiotics (I) is a multistep reaction [I]. Once formed, the equimolar and inactive complexes EI give rise to modified complexes EI* which, in turn, undergo breakdown with the concomitant release of the reactivated enzyme (E) and of a modified and inactive antibiotic molecule (X). Depending upon the antibiotic, the EI* complexes have at 37 "C long half-lives of 40 -11 000 min. The rates of formation of complexes EI*, as determined by measuring the quenching of the fluorescence of the R61 enzyme in the presence of large amounts of antibiotics, suggested that the simplest mechanism which accounted for all the experimental results so far accumulated, was :where the formation of complex EI was a rapid equilibrium process [I]. Earlier studies [2] had shown that by incubating together the R61 enzyme, the tripeptide AC,-L-LYS-D-Ala-D-Ala donor and a p-lactam antibiotic for some time (30 -60 min) at 37 "C and on the basis of the residual activity measured under these conditions, Lineweaver-Burk plots were obtained according to which the R61 enzyme was competitively inhibited by the antibiotics with regard to the peptide donor Eur. J. Biochem. 57 (1975) (DD-carboxypeptidase activity: Ac,-L-Lys-D-Ala-D-The enzyme used in these experiments had been purified to protein homogeneity; the antibiotic concentrations were much higher than that of the enzyme and the amount of hydrolysis product formed never exceeded 15% of the original amount of substrate. Because of this low utilization of substrate, it was assumed that the experiments had been carried out under conditions of initial velocity. On the basis of the model proposed above for the interaction between the enzyme and penicillin, however, it appears that this assumption was not justified. Indeed, the estimation of the velocity of the formation of complex EI* showed that the concentration of free enzyme and hence the velocity of the reaction continuously decreased throughout the incubation of the reaction mixture. It thus became essential to examine whether or not the 'competitive' inhibition (in Lineweaver-Burk plots) was compatible with the mechanisms proposed for the interaction between the R61 enzyme and penicillin. THEORETICAL BACKGROUND SymbolsThe symbols are those used previously [I]. In addition, S = substrate (Ac,-L-Lys-D-Ala-D-Ala) ; [S] = substrate concentration; P = ...

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Section: Discussioncontrasting

confidence: 89%

Section: With Each Of These Antibiotics the Conditions [El @ [I] Andmentioning

confidence: 68%

Section: With Each Of These Antibiotics the Conditions [El @ [I] Andmentioning

confidence: 99%

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Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics

Frère

Ghuysen

Perkins

1975

European Journal of Biochemistry

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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].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.…”

mentioning

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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“…the completely reactivatable enzyme with the lower penicillin sensitivity. D-Alanyl-D-alanine carboxypeptidase (DD-carboxypeptidase), peptidoglycan transpeptidase and peptidoglycan endopeptidase are membrane-bound enzymes engaged in the final stages of bacterial peptidoglycan synthesis and have been identified as specific targets for the inhibitory action of penicillins and cephalosporins (for review see [1,2], also [ 3 -81). During normal synthesis of peptidoglycan in Escherichiu coli, transpeptidase and DD-carboxypeptidase appear to carry out concerted activities.…”

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confidence: 99%

d‐Alanyl‐d‐Alanine Carboxypeptidase in the Bacterial Form and L‐Form ofProteus mirabilis

Martin

Maskos

Bürger

1975

European Journal of Biochemistry

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Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki = 4 x M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 x M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial m-carboxypeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. rnirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.D-Alanyl-D-alanine carboxypeptidase (DD-carboxypeptidase), peptidoglycan transpeptidase and peptidoglycan endopeptidase are membrane-bound enzymes engaged in the final stages of bacterial peptidoglycan synthesis and have been identified as specific targets for the inhibitory action of penicillins and cephalosporins (for review see [1,2], also [ 3 -81). During normal synthesis of peptidoglycan in Escherichiu coli, transpeptidase and DD-carboxypeptidase appear to carry out concerted activities. Transpeptidase forms a sufficient number of cross linkages between peptide side chains of the polymer, DD-Carboxypeptidase, on the other hand, seems to be required for splitting off the C-terminal D-alanine from the many (two thirds of the total number) precursor pentapeptide side chains -L-Ala-D-Glu-rns-A,pm-DAbbreviurions. ms-A,pm, mr.so-2,6-diaminopimelic acid ; UDP- MurNAc-pentapeptide, UDP-N-acetyhnuramyl-L-alanyl-D-glutamyl-(L)-meso-diaminopimelyl-(L)-D-alanyl-D-alanine;Genapol X-100, isotridecanol polyglycol ether (i = 10); Hepes, N-2-hydroxyethylpiperazine-NJ-2-ethane sulfonic acid; Tes, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid.Definition. IDso, the concentration of antibiotic which decreased by 50 the rate of enzyme activity.Enzymes. DD-Carboxypeptidase or D-alanyl-D-alanine carboxypeptidase (EC 3.4.12.6); p-lactamase or penicillinase (EC 3.5.2.6); D-amino acid oxidase (EC 1.4.3.3); peroxidase (EC 1.11.1.7).Ala-D-Ala, which are normally destined to remain uncross linked in the finished peptidoglycan or do not participate in cross linkage with their C-terminal ends [9], thus converting them into tetrapeptides -L-Ala-D-Glu-ms-A,pm-D-Ala.As shown in a cell-free preparation of E. coli inhibition of transpeptidase and DD-carboxypeptidase by b-lactam antibiotics results in the formation of a defective, soluble peptidoglycan with uncrosslink...

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Beam Loss Due to G10 Targeting

Cited by 3 publications

References 0 publications

Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics

Interaction between the Exocellular DD‐Carboxypeptidase‐Transpeptidase from Streptomyces R61, Substrate and β‐Lactam Antibiotics

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

d‐Alanyl‐d‐Alanine Carboxypeptidase in the Bacterial Form and L‐Form ofProteus mirabilis

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