Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

Catalytic cycle of a class A β-lactamase illustrated for a monobactam substrate. All class A β-lactamases employ an active site serine nucleophile to cleave the β-lactam bond of the substrate in a two-step acylation-deacylation reaction cycle that leads to overall hydrolysis. The acylation reaction initiates with the formation of a precovalent substrate complex (stage 1). A general base-catalyzed nucleophilic attack on the β-lactam carbonyl by the serine hydroxyl proceeds through a tetrahedral intermediate (stage 2) to form a transient acyl-enzyme adduct (stage 3). In the deacylation step, the acyl-enzyme adduct (stage 3) undergoes a general base-catalyzed attack by a hydrolytic water molecule to form a second tetrahedral intermediate (stage 4), which then collapses to form a postcovalent product complex (stage 5), from which the hydrolyzed product is released.