Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and β-lactam binding activities

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with β-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k2/K‘ value relative to other PBPs for acylation with penicillin (7.7 × 105 M-1 s-1) at pH 8.5 at 25 °C and hydrolyzed bound antibiotic very slowly (k3 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a kcat of 580 s-1 and a kcat/Km of 1.8 × 105 M-1 s-1 with the substrate Nα-Boc-Ne-Cbz-l-Lys-d-Ala-d-Ala. This is the highest kcat value yet reported for a PBP or other serine peptidases. Activity against a ∼d-Ala-d-Lac peptide substrate was ∼2-fold lower than against the analogous ∼d-Ala-d-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence pro...