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

Abstract

(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.

Penicillin-binding proteins in bacteria

“…It should be noted that the results of the present study rest on the assumptions that (i) PBPs are independent of growth phase (2); (ii) PBPs are bound to membranes and not to cell walls (as are some autolysins [10]); (iii) PBP-binding activity is not substantially altered by solubilization in 2% Triton X-100 and 1 M NaCl. reported, slow release has been associated with fragmentation of the penicillin molecule (11,13), and fast release has been associated with opening of the f8-lactam ring to give penicilloic acid (17,31). Since both processes are enzymatic, the different modes ofrelease may reflect differences in the architecture of the active site.…”

Penicillin-binding proteins of Streptomyces cacaoi, Streptomyces olivaceus, and Streptomyces clavuligerus

“…Penicillin-binding proteins (PBP) have been studied in many species of bacteria mainly from a standpoint of elucidating the mechanism of action of fl-lactam compounds (2). Some PBP have a weak ,B-lactamase activity (6). These proteins may be targets of penicillins and other ,B-lactam compounds (5,24), and at least some are essential for the survival of bacteria.…”

Interactions between beta-Lactam Antibiotics and Isolated Membranes of Streptococcus faecalis ATCC 9790

“…This compound, however, is quickly hydrolysed to N-formyl-D-penicillamine in aqueous solutions at pH 4-7 and hence the demonstration of its release may be difficult. The formation of phenylacetylglycine by interaction of benzylpenicillin with the DD-carboxypeptidase isolated from the membranes of Bacillus stearothermophilus was demonstrated but the transformation of the other part of the molecule into dimethylthiazoline carboxylic acid was speculative [9].…”