By using steady and transient state #kinetics as well as linear free-energy relationships (#LFER) we show that the breakup of the covalent phosphoryl intermediate – which is the 'lethal' step for acetylcholinesterase – is surprisingly fast in this enzyme.

Instead, the rate-limiting step is the initial formation of the covalent intermediate. This step subsequently gets vastly accelerated through our directed evolution campaign (by increased leaving group stabilisation). (5/6)