Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Weilie Zhang, Qicun Shi, Samy O. Meroueh, Sergei B. Vakulenko, Shahriar Mobashery

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Abstract

Penicillin-binding proteins (PBPs) and β-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 γ-thialysine at each of these positions. The pH dependence of k cat/Km and of kcat for the wild-type PBP 5 and for the two γ-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.

Original languageEnglish (US)
Pages (from-to)10113-10121
Number of pages9
JournalBiochemistry
Volume46
Issue number35
DOIs
StatePublished - Sep 4 2007

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ASJC Scopus subject areas

  • Biochemistry

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