KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate.

摘要: Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem are poorly hydrolyzed most owing to slow hydrolysis intermediate. However, emergence KPC-2 carbapenemase has resulted in widespread resistance these drugs, suggesting it operates more efficiently. Here, we investigated unusual features that enable this resistance. We show 20,000-fold increased rate compared with common TEM-1 β-lactamase. Furthermore, kinetic analysis active site alanine mutants indicates carbapenem is concerted effort involving multiple residues. Substitution Asn170 greatly decreases rate, but residue conserved both and non-carbapenemase β-lactamases, promotes only context KPC-2. X-ray structure determination N170A enzyme complex imipenem suggests may prevent inactivation deacylating 6α-hydroxyethyl substituent carbapenems. In addition, Thr235 residue, which interacts C3 carboxylate carbapenems, also contributes strongly reaction. contrast, mutation Arg220 Thr237 residues acylation and, paradoxically, improves binding affinity for Thus, role be ground state destabilization enzyme-substrate or, alternatively, ensure proper alignment substrate key catalytic facilitate acylation. These findings suggest modifications scaffold avoid