title: Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis.
abstract: The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.
authors.affiliation: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: F
authors.initials: F
authors.lastname: Faustinella
authors.forename: L C
authors.initials: LC
authors.lastname: Smith
authors.forename: C F
authors.initials: CF
authors.lastname: Semenkovich
authors.forename: L
authors.initials: L
authors.lastname: Chan
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000235:genetics; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D012694:Serine; D004247:DNA; D008071:Lipoprotein Lipase