Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli.
This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002268:Carboxypeptidases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010458:Peptidyl Transferases; D014357:Trypsin
D000596:Amino Acids; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010446:Peptide Fragments; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin; D010400:Penicillin G