PMID:17685588

Citation	Zhang, W, Shi, Q, Meroueh, SO, Vakulenko, SB and Mobashery, S (2007) Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli. Biochemistry 46:10113-21
Abstract	Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.
Links	PubMed Online version:10.1021/bi700777x
Keywords	Acylation; Amino Acid Substitution; Binding Sites; Catalysis; Cell Wall/chemistry; Cell Wall/metabolism; Cysteine/analogs & derivatives; Cysteine/chemistry; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Hydrogen-Ion Concentration; Lysine/chemistry; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Penicillin-Binding Proteins/chemistry; Penicillin-Binding Proteins/metabolism; Peptidoglycan; Protein Binding; Proton Pumps; beta-Lactamases/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:DACA		GO:0009002: serine-type D-Ala-D-Ala carboxypeptidase activity	ECO:0000314:		F		DD carboxypeptidase activity was demonstrated with a fluorescent assay. Figure 3 demonstrates pH profiling and optimal PBP5 catalysis.	complete CACAO 8328
ECOLI:DACA	enables	GO:0009002: serine-type D-Ala-D-Ala carboxypeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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