Catalytic Mechanism of Penicillin-Binding Protein 5 of Escherichia coli
| Autor: | Sergei B. Vakulenko, Samy O. Meroueh, Shahriar Mobashery, Qicun Shi, Weilie Zhang |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Models
Molecular Penicillin binding proteins Stereochemistry Acylation Peptidoglycan medicine.disease_cause Biochemistry Catalysis beta-Lactamases chemistry.chemical_compound Residue (chemistry) Cell Wall Escherichia coli medicine Penicillin-Binding Proteins Cysteine Enzyme kinetics Binding site chemistry.chemical_classification Binding Sites biology Escherichia coli Proteins Lysine Active site Hydrogen-Ion Concentration Proton Pumps Enzyme Amino Acid Substitution Models Chemical chemistry Mutagenesis Site-Directed biology.protein Protein Binding |
| Zdroj: | Biochemistry. 46:10113-10121 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi700777x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|