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.
Highlights
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The relatedness was strongly suggested,Penicillin and other p-lactam antibiotics exert their lethal effect by binding to and covalently inactivating the enzymes that are involved in the synthesis of peptidoglycan at the bacterial cell surface [1].These cell wall synthesis enzymes, called penicillin-binding proteins (PBPs),’ canbe detected by incubation of bacterial membranes with radiolabeled penicillin G, followedby SDS-polyacrylamide gel electrophoresis and fluorography [2]
Box VII Sequences of PBPs and Site-saturation Mutagenesis-Alignment of the sequences of E. coli PBPs, the Streptomyces R61 DD-peptidase, and several p-lactamases in the region of Box VI1 shows the conserved character of the triad (Fig. 1).This triad can be KTG, KSG, or HTG
Summary
Q 1992 by The American Society for Biochemistry and Molecular BiologyI,nc. Vol 267, Na. 16, Issue of June 5, pp. 11386-11391.1992 Printed in U.S.A. The evolutionary relationship between these two groups of penicillin-interactive enzymes was further investigated using homology searches andamino acid alignments, using the StreptomycesR61 D-alanyl-D-alaninepeptidase as areference, of all of the known sequences of PBPs and 8-lactamases[15]. This method identified several regions, called boxes, that consist of strict identities or homologous amino acids. To catalyze the acyl-exchange reactions that occur during turnover One of these conserved regions, called Box 11, is the well-characterized Ser-X-X-Lys tetrad that contains the active-site serine nucleophile. We chose the method of site-saturationmutagenesis to investigate the role of the basic residue inthe Box VI1 region of a solubleform of PBP 5 in which Lys213 was mutated to 18 other amino acids and the properties of these mutants were investigated
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