pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

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• 作者：Stefanova ; Miglena E. ; Davies ; Christopher ; Nicholas ; Robert A. ; Gutheil ; William G.
• 关键词：Penicillin-binding protein ; Escherichia coli ; pH profile ; pK_a ; Substrate specificity ; Enzyme inhibition ; Ac ; acetyl ; Ac₂-KAA ; diacetyl-l-Lys-d-Ala-d-Ala (Ac₂-l-Lys-d-Ala-d-Ala) ; Alk ; alkylated ; AMPSO ; 3-([1 ; -dimethyl-2-hyd
• 刊名：Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology
• 出版年：2002
• 出版时间：June 3, 2002
• 年：2002
• 卷：1597
• 期：2
• 页码：292-300
• 全文大小：192 K

文摘

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure–function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five α- and ?-substituted l-Lys-d-Ala-d-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6–12.3, and the k_cat/K_m vs. pH profile for activity against Ac₂-l-Lys-d-Ala-d-Ala was bell-shaped, with pK_as at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed dd-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A β-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.