Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222).
The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000302:isolation & purification*; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010458:Peptidyl Transferases; D014357:Trypsin
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010446:Peptide Fragments; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin