Despite advances in structural and computational methods it remains a challenge to predict mutations that will lead to increased catalytic activity. The evolution of insecticide resistance provides an amenable eukaryotic model system for exploring enzyme evolution. Many insect species have evolved resistance to organophosphate (OP) insecticides through catalytic detoxification of OP-insecticides. We have investigated the evolution of an OP-hydrolase (αE7) from the blowfly Lucilia cuprina. In nature a Gly137Asp mutation in αE7 results in a qualitative change to the catalytic mechanism, with Asp137 acting as a general base to activate a nucleophilic water molecule for dephosphorylation of the active site serine. However, this new active site is disordered, and only samples the catalytically productive geometry a fraction of the time. We have directed the evolution of αE7 towards greater OP-hydrolase activity, resulting in an >1000-fold increase in the rate of OP-hydrolysis, potentially through stabilization of the most optimal active site geometry. Our results suggest that mutations that stabilize pre-existing conformational sub-states may be important for the evolution of enhanced catalytic activity. Concerningly, our directed evolution data indicate that the Gly137Asp mutation could be the first of many steps toward efficient OP-insecticide degradation.