Fluorescence and circular dichroism studies on the Streptomyces R61 DD-carboxypeptidase-transpeptidase. Penicillin binding by the enzyme

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 ▲]

Streptomyces DD-carboxypeptidases as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors

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 ▲]

Exocellular beta-lactamases of Streptomyces albus G and strains R39 and K11

in Antimicrobial Agents and Chemotherapy (1973), 3(2), 289-298

The beta-lactamases excreted by the highly benzylpenicillin-susceptible Streptomyces strain R39 and the highly benzylpenicillin-resistant Streptomyces albus G were isolated and purified. Neither beta ... [more ▼]

The beta-lactamases excreted by the highly benzylpenicillin-susceptible Streptomyces strain R39 and the highly benzylpenicillin-resistant Streptomyces albus G were isolated and purified. Neither beta-lactamase exhibited dd-carboxypeptidase activity. Both were anionic at pH 8.3, did not require metal ions, and were not sensitive to iodine, but were inhibited by Cu(2+) and readily inactivated by heat. p-Chloromercuribenzoate, iodoacetate, p-aminobenzoate, and substrates and inhibitors of dd-carboxypeptidase had no effect on beta-lactamase activity. The K(m) and V(max) values for beta-lactamase activity were studied with 6-aminopenicillanic acid and with various penicillins and cephalosporins. The beta-lactamase from the related strain K11 of Streptomyces, which is intermediate in its susceptibility to benzylpenicillin, was partially purified, and its activity was compared on the various substrates. [less ▲]

DD-carboxypeptidases/Transpeptidases and Penicillin Action

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

Exocellular and membrane-bound transpeptidases in Streptomyces

in 1st International Congress for Bacteriology, Jerusalem, 2-7 September, 1973. Abstracts. Vol. II. Free papers. (1973)

Structure of the wall peptidoglycan of streptomyces R39 and the specificity profile of its exocellular DD-carboxypeptidase-transpeptidase for peptide acceptors

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 ▲]

Penicillin-sensitive DD-carboxypeptidases from Streptomyces strains R39 and K11

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

The Streptomyces DD-carboxypeptidase-transpeptidase system as a model for the study of penicillin action

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 ▲]

Wall peptidoglycan in Aerococcus viridans strains 201 Evans and ATCC 11563 and in Gaffkya homari strain ATCC 10400

in Biochemistry (1971), 10(11), 2170-2175

DD-carboxypeptidases and Mechanism of Action of Penicillin

in Muñoz, E.; García-Ferrandiz, F.; Vazquez, D. (Eds.) Molecular mechanisms of antibiotic action on protein biosynthesis and membranes. Proceedings of a symposium held at the University of Granada (Spain) June 1st-4th (1971)