Mutation of the catalytic site Asp177 to Glu177 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, Asp177 in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp177 to Glu177 produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp177 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 Asp177 and Asp206, another acidic residue that could participate in the catalytic triad. The Glu177 mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp177. Several mutants of Asp206 were normally active, thus, confirming the important role of Asp177 in pancreatic lipase function. Additionally, we found that the Glu177 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.
Lowe
M E
ME
Department of Pediatrics, Washington University School of Medicine at St. Louis Children's Hospital, MO 63110, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics*; D006801:Humans; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D011489:Protein Denaturation; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection; D014508:Urea
D001224:Aspartic Acid; D018698:Glutamic Acid; D014508:Urea; D008049:Lipase; D010450:Endopeptidases