Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.
All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.
Malhotra
K T
KT
Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
Nicholas
R A
RA
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D001426:Bacterial Proteins; D002268:Carboxypeptidases / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005822:Genetic Vectors; D006602:Hexosyltransferases; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010458:Peptidyl Transferases; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D001120:Arginine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; C019632:alanine carboxypeptidase; D002267:Muramoylpentapeptide Carboxypeptidase; D008239:Lysine