Ion pairing between lipase and colipase plays a critical role in catalysis

Among the polar interactions occurring in pancreatic lipase/colipase bindin g, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on colipase has been described. These residues are strictly conserved among s pecies, suggesting that the ion pair is likely to play an important role. T herefore, in order to prevent this interaction, mutations intended to neutr alize or inverse the charge of these residues have been introduced in the c DNAs encoding horse lipase and colipase, The recombinant proteins have been expressed in insect cells, and their catalytic properties have been invest igated. In all cases, preventing the formation of the correct ion pair Lys( 400)/Glu(45) leads to lipase-colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt mic elles, Diethyl p-nitrophenyl phosphate inhibition experiments with either m utant lipase or mutant colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-colipase-micelle ternary complex by cont ributing to a correct orientation of colipase relative to lipase resulting in a proper opening of the flap.