title: Bacillus cereus 569/H penicillinase serine-44 acylation by diazotized 6-aminopenicillanic acid.
abstract: Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.
mesh_terms: D000215:Acylation; D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D001409:Bacillus cereus / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D013058:Mass Spectrometry; D010397:Penicillanic Acid / Q000494:pharmacology* / Q000031:analogs & derivatives; D010405:Penicillinase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012694:Serine; D013171:Spores, Bacterial / Q000201:enzymology; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D012694:Serine; D010397:Penicillanic Acid; D010405:Penicillinase; D001618:beta-Lactamases; C004408:aminopenicillanic acid