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 incorpor ates a serine-histidine-acidic group triad. In general, the acidic gro up is an aspartate, Asp(177) in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-speci fic mutagenesis of Asp(177) to Glu(177) produced a mutant human pancre atic lipase with near normal activity against triolein, thereby, raisi ng questions about the role of Asp(177) in the catalytic triad and abo ut the evolutionary pressure which selected Asp over Glu in the cataly tic mechanism. To address these questions, we constructed and expresse d mutants of Asp(177) and Asp(206), another acidic residue that could participate in the catalytic triad. The Glu(177) mutant had a substrat e specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp(177). Sev eral mutants of Asp(206) were normally active, thus, confirming the im portant role of Asp(177) in pancreatic lipase function. Additionally, we found that the Glu(177) mutant had increased susceptibility to prot eases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanat ion for the preference of aspartate in the catalytic triad of human pa ncreatic lipase.