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

M.E Lowe

doi:10.1016/s0021-9258(18)41893-5

Abstract

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 Ser153, His264, 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 Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.

Highlights

In this study, theessential serine residue and2 other glassbeads (4).Activity against water-soluble substrates, such aminoacids in human pancreatic triglyceride lipase as p-nitrophenyl phosphate, was not affected by modifying were tested the essential serine
When crystals of the human ing glutamicacidforproducedan active protein. lipase were soaked in a solution of a reversible inhibitor, These results demonstrate that SerIiss3involved in the butylboronic acid, the difference electron density map located catalytic site of pancreaticlipase and is not crucial for the inhibitor near the hydroxyl group of the essential serine, interfacial binding.the essential roles of indicating that theresidue is at or near the catalytic site
In addition to the studiescited previously and the data presented here, several other lines of evidence support the conclusion that S e P participatesin the catalytic site

Summary

The CatalyticSite Residues and Interfacial Binding oHf uman Pancreatic Lipase*

Theessential serine residue and other glassbeads (4).Activity against water-soluble substrates, such aminoacids in human pancreatic triglyceride lipase as p-nitrophenyl phosphate, was not affected by modifying (triacylglycerolacylhydrolase,EC 3.1.1.3) were tested the essential serine These results suggested that the serine for their contribution to the enzyme’csatalytic site or was in the lipid-binding domain and not the catalytic site. An intriguing aspect of pancreatic lipase catalysis is the Manipulations of DNA were done by standard methods (8).Transremarkable and substantial increase in activity when insoluble surfaces are present (2) This property suggests an additionalstepinthe sequence of reactions that lead tothe hydrolysis of triglycerides (3). The strate is bound at the catalytic site This model predicts the sequence for the human pancreatic cDNA was previously reported presence of separate sitesfor interfacial binding and catalysis. The DNA for transfection was purified on Qiagen columns as described by the manufacturer

RESULTS

Pancreatic Lipase Catalytic Site

DISCUSSION