The inhibitors bind covalently with the enzymes and indicate those which are irreversible. Formation of covalent link between irreversible inhibitor and an enzyme is common. Irreversible inhibitors are another useful tool for studying reaction mechanisms. For example, iodoacetate is an irreversible inhibitor to enzymes like papain and glyceraldehyde-3-phosphate dehydrogenase. It combines with sulfhydryl (SH) groups at active site of three enzymes and makes them inactive.

Diisopropyl fluorophosphates (DFP) is a nerve gas developed by Germans during the Second World War. DFP irreversibly bind with enzymes containing serine at the active site of acetylcholine esterase (essential for nerve condition) and results in paralysis of vital body function.