PMID:1597468
| Citation |
Malhotra, KT and Nicholas, RA (1992) Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding. J. Biol. Chem. 267:11386-91 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Arginine/genetics; Bacterial Proteins; Carboxypeptidases/metabolism; Carrier Proteins/genetics; Escherichia coli/enzymology; Escherichia coli/metabolism; Genetic Vectors; Hexosyltransferases; Hydrolysis; Kinetics; Lysine/genetics; Molecular Sequence Data; Muramoylpentapeptide Carboxypeptidase/genetics; Mutagenesis, Site-Directed; Penicillin-Binding Proteins; Penicillins/metabolism; Peptidyl Transferases; Sequence Alignment |
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