Mutation of the catalytic site Asp(177) to Glu(177) in human pancreatic lipase produces an active lipase with increased sensitivity to proteases

The catalytic mechanism for members of the lipase gene family incorporates a serine-histidine-acidic group triad. In general, the acidic group is an aspartate, Asp(177) in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp(177) to Glu(177) produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp(177) in the catalytic triad and about the evolutionary pressure which selected Asp over Glu in the catalytic mechanism. To address these questions, we constructed and expressed mutants of Asp(177) and Asp(206), another acidic residue that could participate in the catalytic triad. The Glu(177) mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp(177). Several mutants of Asp(206) were normally active, thus, confirming the important role of Asp(177) in pancreatic lipase function. Additionally, we found that the Glu(177) mutant had increased susceptibility to proteases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanation for the preference of aspartate in the catalytic triad of human pancreatic lipase.