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The catalytic site residues and interfacial binding of human pancreatic lipase.

1992; Elsevier BV; Volume: 267; Issue: 24 Linguagem: Inglês

10.1016/s0021-9258(18)41893-5

1083-351X

Mark E. Lowe,

Diabetes Management and Research

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3)were tested for their contribution to the enzyme's catalytic site or interfacial binding site.By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at SerIs3, HisZB4, and Asp177.The mutant cDNAs were expressed in transfected COS-1 cells.Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis.The activity of the expressed proteins against triolein and the interfacial binding was measured.Proteins with mutations in S e P 3 were secreted by the cells and bound to interfaces but had no detectable activity.Changing HisZB4 to a leucine or to an asparagine also produced inactive lipase.Substituting glutamic acid for produced an active protein.These results demonstrate that SerIs3 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding.Moreover, the essential roles of HisZB4 and Asp'77 in catalysis were demonstrated.A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.Triglycerides comprise 95% of the fats in the Western diet.For efficient uptake by enterocytes, triglycerides must be hydrolyzed into free fatty acids and monoacylglycerides.Pancreatic lipase (EC 3.1.1.3)is the predominant enzyme required for efficient digestion of triglycerides (1).Pancreatic lipase is synthesized and secreted by the pancreas and requires another pancreatic exocrine protein, colipase, for full activity under physiological conditions (2).An intriguing aspect of pancreatic lipase catalysis is the remarkable and substantial increase in activity when insoluble surfaces are present (2).This property suggests an additional step in the sequence of reactions that lead to the hydrolysis of triglycerides (3).The enzyme must bind to the surface of the substrate emulsion before a molecule of substrate is bound at the catalytic site.This model predicts the presence of separate sites for interfacial binding and catalysis.Attempts to identify amino acids involved in catalysis and interfacial binding have produced conflicting results.Reaction of porcine pancreatic lipase with diethyl p-nitrophenyl phosphate modified a single serine and abolished both lipase activity against micellar substrates and binding to siliconized