Binding of [14C]Penicillin G to the Membrane-bound and the Purified d-Alanine Carboxypeptidases from Bacillus stearothermophilus and Bacillus subtilis and Its Release

Abstract [14C]Penicillin G bound to the Bacillus subtilis and Bacillus stearothermophilus d-alanine carboxypeptidases in either the membrane-bound or purified states. This binding was subsequently reversed with a half-time ranging from 10 min to several days, depending on the enzyme, the penicillin derivative, and the temperature. The released material was neither intact penicillin nor penicilloic acid but rather some other penicillin derivative. The enzymes were thus formally penicillinases of low activity, although they were not β-lactamases. In addition to the release catalyzed by the native enzyme, hydrolysis of the penicillin-enzyme complex also occurred under certain circumstances after enzyme denaturation. Prior to release from the enzyme, the penicillin appeared to be covalently bound. It was not released by boiling sodium dodecyl sulfate, by 8 m urea, or by 6 m guanidine HCl. Likewise, intact penicillin was not freed by digestion with pronase; the material released upon digestion was a mixture of penicilloic acid and an unidentified compound, possibly penicillin-peptide.