Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241

J Biol Chem. 1992 Feb 25;267(6):4161-5.

Abstract

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Asparagine / metabolism
  • Binding Sites
  • Blotting, Northern
  • Catalysis
  • Cells, Cultured
  • Chromatography, Affinity
  • DNA / genetics
  • Histidine / metabolism
  • Humans
  • Kidney / cytology
  • Kidney / embryology
  • Kidney / enzymology
  • Lipoprotein Lipase / genetics
  • Lipoprotein Lipase / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleic Acid Hybridization
  • Plasmids
  • RNA / genetics
  • Serine / metabolism

Substances

  • Serine
  • Histidine
  • RNA
  • Asparagine
  • DNA
  • Lipoprotein Lipase