Indirect-acting cholinergic agonists work by interacting with an enzyme called acetylcholinesterase (AChE) in the synaptic cleft. They can be reversible or irreversible inhibitors and have different effects on the enzyme.

Reversible inhibitors like edrophonium bind to a specific part of the enzyme called the anionic catalytic site. They form noncovalent bonds, which means they are not strongly attached to the enzyme. This creates a temporary and less stable enzyme–inhibitor complex, leading to a short duration of action.

Other reversible inhibitors, such as carbamic acid esters, bind to a different part of the enzyme called the esteratic site. They replace a specific group on the enzyme with their carbamyl group. This creates a carbamoylated enzyme, which undergoes slow hydrolysis over several minutes. As a result, these inhibitors have a longer duration of action.

Irreversible inhibitors, such as organophosphates, also bind to the esteratic site of the enzyme. However, they form covalent bonds, which are strong and permanent. These inhibitors phosphorylate a specific group on the serine hydroxyl enzyme, creating an irreversible enzyme–inhibitor complex that lasts for a long time.

The stability of the irreversible enzyme–inhibitor complex can be further enhanced through a process called aging. During aging, one of the bonds between oxygen and phosphorus in the inhibitor breaks, making the phosphorus–enzyme bond even stronger.