title: Human hepatic and lipoprotein lipase: the loop covering the catalytic site mediates lipase substrate specificity.
abstract: Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes that mediate the hydrolysis of triglycerides (TG) and phospholipids (PL) present in circulating plasma lipoproteins. Relative to triacylglycerol hydrolysis, HL displays higher phospholipase activity than LPL. The structural basis for this difference in substrate specificity has not been definitively established. We recently demonstrated that the 22-amino acid loops ("lids") covering the catalytic sites of LPL and HL are critical for the interaction with lipid substrate (Dugi, K.A., Dichek, H.L., Talley, G.D., Brewer, H.B., Jr., and Santamarina-Fojo, S. (1992) J. Biol. Chem. 267, 25086-25091). To determine whether the lipase lid plays a role in conferring the different substrate specificities of HL and LPL, we have generated four chimeric lipases. Characterization of these chimeric enzymes using TG (triolein and tributyrin) or PL (dioleoylphosphatidylcholine (DOPC) vesicles, DOPC proteoliposomes, and DOPC-mixed liposomes) substrates demonstrated marked differences between their relative PL/TG hydrolyzing activities. Chimeric LPL containing the lid of HL had reduced triolein hydrolyzing activity (49% of the wild type), but increased phospholipase activity in DOPC vesicle, DOPC proteoliposome, and DOPC-mixed liposome assay systems (443, 628, and 327% of wild-type LPL, respectively). In contrast, chimeric HL containing the LPL lid was more active against triolein (123% of the wild type) and less active against DOPC (23, 0, and 30%, respectively) than normal HL. Similar results were obtained when the lipase lids were exchanged in chimeric enzymes containing the NH2-terminal end of LPL and the COOH-terminal domain of HL. Exchange of the LPL and HL lids resulted in a reversal of the phospholipase/neutral lipase ratio, establishing the important role of this region in mediating substrate specificity. In summary, the lid covering the catalytic domains in LPL and HL plays a crucial role in determining lipase substrate specificity. The lid of LPL confers preferential triglyceride hydrolysis, whereas the lid of HL augments phospholipase activity. This study provides new insight into the structural basis for the observed in vivo differences in LPL and HL function.
authors.affiliation: Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Dugi
authors.forename: H L
authors.initials: HL
authors.lastname: Dichek
authors.forename: S
authors.initials: S
authors.lastname: Santamarina-Fojo
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
chemical_list: D008074:Lipoproteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D008049:Lipase; D008071:Lipoprotein Lipase