Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.
Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents.
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