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See detailThe active centres in penicillin-sensitive enzymes
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Philosophical Transactions : Biological Sciences (1980), 289(1036), 285-301

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site ... [more ▼]

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced. [less ▲]

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See detailExocellular DD-carboxypeptidases-transpeptidases from Streptomyces
Frère, Jean-Marie ULg; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg et al

in Methods in Enzymology (1976), 45

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See detailThe penicillin receptor in Streptomyces
Ghuysen, Jean-Marie ULg; Leyh-Bouille, M.; Frère, Jean-Marie ULg et al

in Annals of the New York Academy of Sciences (1974), 235

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See detailKinetics of concomitant transfer and hydrolysis reactions catalysed by the exocellular DD-carboxypeptidase-transpeptidase of streptomyces R61
Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg; Perkins, Harnold R. et al

in Biochemical Journal (1973), 135(3), 483-492

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac ... [more ▼]

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac(2)-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac(2)-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac(2)-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested. [less ▲]

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See detailMolecular weight and amino acid composition of the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61
Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg; Perkins, Harnold R. et al

in Biochemical Journal (1973), 135(3), 463-468

A procedure allowing the purification of milligram amounts of the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 to protein homogeneity (95% purity) is described. The isolated ... [more ▼]

A procedure allowing the purification of milligram amounts of the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 to protein homogeneity (95% purity) is described. The isolated protein has a molecular weight of about 38000 and consists of one polypeptide chain. Its amino acid composition is presented. [less ▲]

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See detailFluorescence and circular dichroism studies on the Streptomyces R61 DD-carboxypeptidase-transpeptidase. Penicillin binding by the enzyme
Nieto, Manuel; Perkins, Harnold R.; Frère, Jean-Marie ULg et al

in Biochemical Journal (1973), 135(3), 493-505

The circular dichroism of the dd-carboxypeptidase-transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far ... [more ▼]

The circular dichroism of the dd-carboxypeptidase-transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far u.v. it shows negative extrema at 217-218 and 208nm, crossover at 202nm and a positive maximum at about 194nm. The u.v. absorption of the enzyme shows it to contain tyrosine and tryptophan in approx. 3.4:1 ratio. The enzyme is fluorescent with a maximum emission at 318-320nm. The near-u.v. circular dichroism of the protein is extensively affected by binding of penicillin G, but the far u.v. is unaffected. Binding of the antibiotic also causes quenching of the fluorescence of the enzyme. The latter effect has been used to study the binding of penicillin G to the enzyme and the influence exerted upon it by salts, denaturants and peptide substrates and inhibitors. High-affinity binding of penicillin appears to be comparatively slow and reversible, and can occur under conditions in which the protein is enzymically inactive. The thermal denaturation of the enzyme in guanidinium chloride at pH7 is affected by binding of the antibiotic. The presence of even large concentrations of beta-mercaptoethanol neither impaired the activity of the enzyme nor prevented its inhibition by penicillin G or cephalosporin C. A new hypothesis for the molecular mechanism of the interaction of the enzyme with penicillin is proposed. [less ▲]

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See detailStreptomyces DD-carboxypeptidases as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors
Perkins, Harnold R.; Nieto, Manuel; Frère, Jean-Marie ULg et al

in Biochemical Journal (1973), 131(4), 707-718

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a ... [more ▼]

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a whole range of amino acids, peptides and structurally related amino compounds were tested for acceptor function. No compound tested was an acceptor for the enzyme from strain albus G whereas the enzymes from strains R61 and K11 could utilize with varying efficiency a wide range of substances including peptides with N-terminal glycine or d-alanine, omega-amino acids, aminohexuronic acids, 6-aminopenicillanic acid and d-cycloserine. Certain peptides, when present in higher concentration, inhibited the transpeptidation observed at lower concentration. The enzyme from strain R39 would not use any dipeptide as an acceptor, but a few compounds that were not glycine or alpha-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid. [less ▲]

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See detailDD-carboxypeptidase-transpeptidase and killing site of beta-lactam antibiotics in Streptomyces strains R39, R61, and K11
Dusart, Jean; Marquet, Alberto; Ghuysen, Jean-Marie ULg et al

in Antimicrobial Agents and Chemotherapy (1973), 3(2), 181-187

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of beta ... [more ▼]

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of beta-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, beta-lactamase activity was not involved in susceptibility to beta-lactam antibiotics. [less ▲]

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See detailPeptide inhibitors of Streptomyces DD-carboxypeptidases
Nieto, Manuel; Perkins, Harnold R.; Leyh-Bouille, Mélina et al

in Biochemical Journal (1973), 131(1), 163-171

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 ... [more ▼]

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms. [less ▲]

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See detailDD-carboxypeptidases/Transpeptidases and Penicillin Action
Perkins, Harold R; Ghuysen, Jean-Marie ULg; Nieto, Manuel et al

in 1st International Congress for Bacteriology, Jerusalem, 2-7 September, 1973. Abstracts. Vol I. Symposia (1973)

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See detailBacterial Peptidoglycans in Relation to the Membrane and the Mechanism of Action of Penicillin
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Perkins, Harold-R. et al

in Vanek, Z.; Hostalek, Z.; Cudlin, J. (Eds.) Genetics of industrial microorganisms. Bacteria (1973)

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See detailStructure of the wall peptidoglycan of streptomyces R39 and the specificity profile of its exocellular DD-carboxypeptidase-transpeptidase for peptide acceptors
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Campbell, James N. et al

in Biochemistry (1973), 12(7), 1243-1251

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the ... [more ▼]

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl2 buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10−6, 1.5×10−6 and 0.63×10−6s−1 (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [14C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [14C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10−5s−1 and 0.8×10−4s−1 respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site. [less ▲]

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See detailPenicillin-sensitive DD-carboxypeptidases from Streptomyces strains R39 and K11
Leyh-Bouille, Mélina; Nakel, Marlies; Frère, Jean-Marie ULg et al

in Biochemistry (1972), 11(7), 1290-1298

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See detailTranspeptidase activity of Streptomyces D-alanyl-D carboxypeptidases
Pollock, J. J.; Ghuysen, Jean-Marie ULg; Linder, R. et al

in Proceedings of the National Academy of Sciences of the United States of America (1972), 69(3), 662-666

In the presence of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala as donor, and either D-[(14)C]alanine, [(14)C]-glycine, or meso-[(3)H]diaminopimelic acid as acceptor, the DD carboxypeptidases from ... [more ▼]

In the presence of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala as donor, and either D-[(14)C]alanine, [(14)C]-glycine, or meso-[(3)H]diaminopimelic acid as acceptor, the DD carboxypeptidases from Streptomyces R61 and R39 catalyze a transpeptidation reaction with the release of terminal D-alanine from the donor and the formation of either N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-[(14)C]Ala, N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-[(14)C] Gly, or N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-meso- [(3)H]diaminopimelic acid. The reaction appears to be a true transpeptidation, and is not simply a "reversal of hydrolysis". Transpeptidation is inhibited by pencillin at concentrations that inhibit hydrolysis (carboxypeptidase action) of the donor peptide. There are differences in the specificity profiles of the Streptomyces enzymes for acceptor molecules:only the R61 enzyme used [(14)C]Gly-Gly as acceptor; transfer of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala to this acceptor resulted in the formation of N(alpha),N(epsilon)-diacetyl-Lys-D-Ala-[(14)C] Gly-Gly, with the synthesis of a (D-Ala-Gly) peptide bond in an endoposition. [less ▲]

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See detailThe Streptomyces DD-carboxypeptidase-transpeptidase system as a model for the study of penicillin action
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Frère, Jean-Marie ULg et al

in Pratesi, P. (Ed.) Medicinal Chemistry : Special contributions - Milan 1972 (1972)

A new model for the transpeptidation reaction involved in the biosynthesis of the bacterial wall peptidoglycan and for its inhibition by penicillin is proposed. This model is in open conflict with the ... [more ▼]

A new model for the transpeptidation reaction involved in the biosynthesis of the bacterial wall peptidoglycan and for its inhibition by penicillin is proposed. This model is in open conflict with the hypotheses previously postulated. It rests upon the demonstration that 1) carboxypeptidase and transpep-tidase activities are performed by the same enzyme, 2) inhibition of both activities by penicillin is carried out in the absence of irreversible acylation of the protein, 3) the enzyme contains multiple sites some of which are involved in regulation, 4) penicillin does not act as a structural analogue of the donor peptide involved in transpeptidation but may act at the level of regulatory site(s). [less ▲]

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See detailPenicillin-sensitive DD-carboxypeptidase from Streptomyces strain R 61
Leyh-Bouille, Mélina; Coyette, Jacques; Ghuysen, Jean-Marie ULg et al

in Biochemistry (1971), 10(11), 2163-2170

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See detailOn the Streptomyces albus G DD carboxypeptidase mechanism of action of penicillin, vancomycin, and ristocetin
Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg; Nieto, Manuel et al

in Biochemistry (1970), 9(15), 2971-2975

The activity of the D-alanyl-D carboxypeptidase from the penicillin-resistant Streptomyces albus G is not or very little affected by penicillins and related antibiotics. The molecular basis for the ... [more ▼]

The activity of the D-alanyl-D carboxypeptidase from the penicillin-resistant Streptomyces albus G is not or very little affected by penicillins and related antibiotics. The molecular basis for the mechanism of action of penicillin is discussed. The Streptomyces albus G D-alanyl-D carboxypeptidase appears as a model for the study of a mechanism of penicillin resistance that does not involve the enzymatic degradation of the antibiotic. Vancomycin and ristocetin are shown to inhibit the hydrolysis of sensitive peptides by the Streptomyces albus G D-alanyl-D carboxypeptidase and the mechanism of inhibition is discussed. [less ▲]

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See detailIsolation of DD carboxypeptidase from Streptomyces albus G culture filtrates
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Bonaly, Roger et al

in Biochemistry (1970), 9(15), 2955-2961

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See detailSubstrate requirements of the Streptomyces albus G DD carboxypeptidase
Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg; Bonaly, Roger et al

in Biochemistry (1970), 9(15), 2961-2970

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