References of "Perkins, Harold R"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailConformational analysis of peptide substrates and inhibitors of the Zn2+ G and serine R61 D-alanyl-D-alanine peptidases
De Coen, Jean-Louis; Lamotte, Josette ULg; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1981), 121(1), 221-232

The tripeptide Nα,Nɛ-diacetyl-l-lysyl-d-alanyl-d-alanine (Ac2- l-LLys1-dAIa2-dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d-alanyl-d-alanine peptidases, and several ldd tripeptide ... [more ▼]

The tripeptide Nα,Nɛ-diacetyl-l-lysyl-d-alanyl-d-alanine (Ac2- l-LLys1-dAIa2-dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d-alanyl-d-alanine peptidases, and several ldd tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short-range and long-range interactions. Among the many backbone conformers of minimal energy of the øii space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90% or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ∑P values are still as high as 35–50%. All the above tripeptides bind to the serine d-alanyl-d-alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+d-alanyl-d-alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1, 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2-lLys-dAla-dAla show that the main backbone has a tendency to adopt a ring-like shape from which the lLYS side chain protrudes as an extended structure. This latter structure forms with the C-terminal d-alanyl-d-alanine an angle varying between 120° and 180° (depending on the conformers) so that its N-terminal acetyl group is about 1–1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a dAla at position 2 with both d-alanyl-d-alanine peptidases and at position 3 in the case of the serine d-alanyl-d-alanine peptidase. Finally, all the conformers of the lAla2 and lAla3 analogues of Ac2-lLys-dAla-dAla fall outside the backbone conformational space that comprises the φiφi angles exhibited by the four types of conformers of the ldd tripeptides. The lAla2 and lAla3 tripeptide analogues do not bind to the serine d-alanyl-d-alanine peptidase (at least at a 10 mM concentration) but they behave as noncompetitive inhibitors of the Zn2+d-alanyl-d-alanine peptidase. [less ▲]

Detailed reference viewed: 11 (0 ULg)
Full Text
See detailPenicillins and delta-3-cephalosporins as inhibitors and mechanism-based inactivators of D-alanyl-D-Ala peptidases
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Burgen, A. S. V.; Roberts, G. C. K. (Eds.) Topics in molecular pharmacology (1981)

Detailed reference viewed: 9 (0 ULg)
Full Text
Peer Reviewed
See detailThe exchange reaction of peptides R-D-alanyl-D-alanine with D-[14C]alanine to R-D-alanyl-D-[14C]alanine and D-alanine, catalysed by the membranes of Streptococcus faecalis ATCC 9790
Coyette, Jacques; Ghuysen, Jean-Marie ULg; Perkins, Harold R.

in European Journal of Biochemistry (1977), 75(1), 225-229

Under alkaline conditions, the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC 9790 catalyses exchange reactions in which the X-L-R3-D-Ala moiety of peptides of the type X-L-R3-D-Ala-D ... [more ▼]

Under alkaline conditions, the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC 9790 catalyses exchange reactions in which the X-L-R3-D-Ala moiety of peptides of the type X-L-R3-D-Ala-D-Ala is transferred to simple amino compounds such as D-alanine, glycine and glycyl-glycine. The enzyme system is unable, however, to catalyse complex reactions that would simulate the natural transpeptidation reaction. [less ▲]

Full Text
Peer Reviewed
See detailThe peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus. Exocellular, lysozyme-releasable and membrane-bound enzymes.
Leyh-Bouille, Mélina; Dusart, Jean; Nguyen Van, Martine ULg et al

in European Journal of Biochemistry (1977), 81(1), 19-28

The DD-carboxypeptidase (I)-transpeptidase (II) system in Streptomyces strain K15 consists of: (1) a membrane-bound II capable of performing low I activity; and (2) a set of I: (a) membrane-bound, (b ... [more ▼]

The DD-carboxypeptidase (I)-transpeptidase (II) system in Streptomyces strain K15 consists of: (1) a membrane-bound II capable of performing low I activity; and (2) a set of I: (a) membrane-bound, (b) lysozyme-releasable, and (c) exocellular, having low II activities in aq. media and at low acceptor concns. The I are related to each other and may belong to the same pathway leading to enzyme excretion. A similar system occurs in Streptomyces strain R61 except that the membrane-bound I activity is low when compared with the membrane-bound II activity. In S. rimosus, the system consists almost exclusively of the membrane-bound II and the levels of membrane-bound, lysozyme-releasable, and exocellular I are very low. [on SciFinder(R)] [less ▲]

Detailed reference viewed: 24 (2 ULg)
Peer Reviewed
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

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
See detailInteraction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, substrate and beta-lactam antibiotics. A choice of models
Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg; Perkins, Harold R

in European Journal of Biochemistry (1975), 57(2), 353-359

The interaction between the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61 and beta-lactam antibiotics is a multistep process during which a rather stable enzyme - antibiotic complex ... [more ▼]

The interaction between the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61 and beta-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. [less ▲]

Detailed reference viewed: 2 (0 ULg)
Full Text
Peer Reviewed
See detailThe catalytic activity and penicillin sensitivity in the liquid and frozen states of membrane-bound and detergent-solubilised transpeptidase of Streptomyces R61
Dusart, Jean; Marquet, Alberto; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1975), 56(1), 57-65

The Km, app. values of the membrane-bound transpeptidase of Streptomyces R61 for the donor Ac2-L-Lys-D-Ala-D-Ala and the acceptor Gly-Gly are not affected by temperature variations when the reaction ... [more ▼]

The Km, app. values of the membrane-bound transpeptidase of Streptomyces R61 for the donor Ac2-L-Lys-D-Ala-D-Ala and the acceptor Gly-Gly are not affected by temperature variations when the reaction mixtures are incubated in liquid suspensions. At -5 degrees C, the incubation can be carried out either in the liquid or in the frozen state. The enzyme is active in the latter state. In the frozen state, the Km, app. value for the acceptor remains unchanged but there is a 3-fold increase in the maximum velocity, a 10-fold decrease of the Km, app. value for the donor and a 10-fold increase of the benzylpenicillin concentration required to inhibit the enzyme activity by 50% (ID50 value). Temperatures of -35 degrees C or below are required to completely inhibit the membrane-bound enzyme in the frozen state. Cetyltrimethylammonium bromide extracts the transpeptidase both from the isolated membranes and, with a much higher yield, from the intact mycelium. The extracted enzyme is not active in the frozen state, requires detergent for activity, has decreased Km, app. values for both donor and acceptor, exhibits the same sensitivity to benzylpenicillin and cephalosporin C as the membrane-bound transpeptidase (in liquid suspensions) and, like this latter enzyme, has no DD-carboxypeptidase activity. The detergent-extracted transpeptidase penetrates gels of Sephadex-100 and is not sedimented at 200 000 X g. [less ▲]

Detailed reference viewed: 5 (1 ULg)
Full Text
Peer Reviewed
See detailEnzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44
Nguyen-Distèche, Martine ULg; Ghuysen, Jean-Marie ULg; Pollock, Jerry J. et al

in European Journal of Biochemistry (1974), 41(3), 447-455

By using the glutamate-amidated tetrapeptide l-alanyl-d-isoglutaminyl-(l)-meso-diamino-pimelyl-(l)-d-alanine as a probe, there appears to exist in the membranes of Escherichia coli K12 strain 44 a dd ... [more ▼]

By using the glutamate-amidated tetrapeptide l-alanyl-d-isoglutaminyl-(l)-meso-diamino-pimelyl-(l)-d-alanine as a probe, there appears to exist in the membranes of Escherichia coli K12 strain 44 a dd-carboxypoptidase-transpeptidase system which does not recognize this peptide and a dd-carboxypoptidase-transpeptidase system which recognizes it. The dd-carboxypeptidase-endopeptidase system is essentially hydrolytic. It catalyzes the hydrolysis of UDP-N-acetyl-muramyl-pentapeptide into UDP-N-acetylmuramyl-tetrapeptide and the hydrolysis of the wall peptidoglycan peptide dimer into monomers. These activities are not inhibited by the glutamate-amidated tetrapeptide. The system may consist either of two enzyme proteins having predominantly carboxypeptidase activity and endopeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. The dd-carboxypeptidase-transpeptidase system (a) catalyzes concomitant hydrolysis (carboxypeptidase activity) and transfer (natural model transpeptidase activity) reactions with the pentapeptide l-alanyl-γ-d-glutamyl-(l)-meso-diaminopimelyl-(l)-d-alanyl-d-alanine. The transfer reaction leads to the synthesis of a dimer that is identical to the one which occurs in the E. coli wall peptidoglycan; (b) utilizes the glutamate-amidated tetrapeptide as an acceptor. Simultaneous exposure of the pentapeptide and the glutamate-amidated tetrapeptide to the enzyme system leads to the formation of an hybrid monoamidated peptide dimer and causes a decreased hydrolysis of the pentapeptide; (c) by virtue of its own carboxypeptidase activity, it appears to exert some endopeptidase activity. Both carboxypeptidase and endopeptidase activities of this system are inhibited by the glutamate-amidated tetrapeptide, but this represents only a small fraction of the total hydrolytic activity of the membrane Brij-36T extract. (d) The system catalyzes an unnatural model transpeptidation reaction in which glycine replaces d-alanine at the C-terminal position of the nucleotide UDP-N-acetylmuramyl-pentapeptide. This system may also consist either of two enzyme proteins having predominantly natural model transpeptidase activity and unnatural model transpeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. Whatever the exact situation, the E. colidd-carboxypeptidase-transpeptidase system is in many respects, similar to the dd-carboxy-peptidase-transpeptidase single polypeptide enzymes isolated from Streptomyces strains R39 and R61. [less ▲]

Detailed reference viewed: 4 (2 ULg)
Full Text
Peer Reviewed
See detailThe penicillin Target in Bacteria
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Frère, Jean-Marie ULg et al

in Spencer, B. (Ed.) Industrial Aspects of Biochemistry (FEBS Proceedings) (1974)

The bacterial target of beta-lactam antibiotics consists of a set of multiple, membrane-bound receptors. Some of them have been characterized as DD-carboxypeptidases. The DD-carboxypeptidases catalyse the ... [more ▼]

The bacterial target of beta-lactam antibiotics consists of a set of multiple, membrane-bound receptors. Some of them have been characterized as DD-carboxypeptidases. The DD-carboxypeptidases catalyse the opening of amide bonds and transfer the carbonyl carbon to an exogenous nucleophile, and are specifically designed to operate on the D-Ala-D-Ala linkageof L-R-D-Ala-D-Ala terminated peptides (where R is most often a diamino acid residue). The R61, R39 and several Bacilli DD-carboxypeptidases are known to be serine-enzymes and the G DD-carboxypeptidase has been characterized as a metallo (Zn ions) enzyme. Both the R61 and the G enzymes have been crystallized. In turn, the S. faecalis 43,000-Mr DD-carboxypeptidase, which is inhibited by low dose levels of pCMB, might be a thiol-enzyme. The goal pursued is the understanding of the mechanistic properties and functioning of the active centers of the DD-carboxypeptidases at the molecular and atomic levels. The research program involves 1) further characterization of the S. faecalis enzyme (which can be obtained in a water-soluble form); 2) isolation of various Streptomyces membrane-bound enzymes in a truly water-soluble form; 3) sequencing of the G and R61 enzymes; 4) the 2.8 A structure analysis of the G enzyme. (A similar study is conducted by Dr. J.R. Knox at the University of Connecticut, on the R61 enzyme, which enzyme is prepared and purified in this laboratory and then sent to Storrs); 5) conformational studies and quantitative structure activity relationships (QSAR). Source du résumé : http://www.researchcrossroads.org/index.php?view=article&id=50%3Agrant-details&option=com_content&Itemid=64&grant_id=4296130 [less ▲]

Detailed reference viewed: 19 (0 ULg)
Full Text
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)

Detailed reference viewed: 6 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 43 (2 ULg)
Full Text
Peer Reviewed
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

Detailed reference viewed: 6 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 7 (1 ULg)
Full Text
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 ▲]

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
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

Detailed reference viewed: 8 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 14 (1 ULg)
Full Text
Peer Reviewed
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

Detailed reference viewed: 16 (0 ULg)