Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.
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.
Molecular Disease Branch, National Heart Lung and Blood Institute, Bethesda, Maryland 20892.
J
J
Emmerich
O U
OU
Beg
J
J
Peterson
L
L
Previato
J D
JD
Brunzell
H B
HB
Brewer
S
S
Santamarina-Fojo
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D015152:Blotting, Northern; D002384:Catalysis; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012313:RNA / Q000235:genetics; D012694:Serine / Q000378:metabolism
D012694:Serine; D006639:Histidine; D012313:RNA; D001216:Asparagine; D004247:DNA; D008071:Lipoprotein Lipase