Novel site in lipoprotein lipase (LPL415;-438) essential for substrate interaction and dimer stability.
LPL, like other lipases, has the ability to hydrolyze water-insoluble lipid substrates, but the mechanism is incompletely understood. We previously demonstrated a 22-amino acid loop in the amino-terminal domain of LPL to be essential for interaction with lipid substrates (Dugi, K. A., H. L. Dichek, G. D. Talley, H. B. Brewer, Jr., and S. Santamarina-Fojo. 1992. J. Biol. Chem. 267: 25086-25091) and mediation of substrate specificity (Dugi, K. A., H. L. Dichek, and S. Santamarina-Fojo. 1995. J. Biol. Chem. 270: 25396-25401). The carboxy-terminal domain, LPL415-438, contains two highly conserved hydrophobic stretches, and represents a candidate region for substrate interactions. Specific point mutations or deletion of the region between the hydrophobic stretches (LPL419-430) caused up to 90% selective loss of hydrolyzing activity against water-insoluble triolein, but not against water-soluble tributyrin, implicating a crucial function for LPL419-430 in the interaction with lipid substrates. In contrast, mutations introduced into the hydrophobic regions led to concomitant changes in tributyrin and triolein activities. The presence of an additional positive charge at position 416 yielded a gain of function mutant with 3-fold increased activity. This mutant was about three times more stable at 37 degrees C than wild-type LPL, suggesting an important role for the hydrophobic regions in LPL dimer stability. In summary, our data demonstrate that the carboxy-terminal region LPL415-438 plays an important role in both the interaction of LPL with lipid substrates and the stability of the LPL homodimer.
Keiper
T
T
Department of Internal Medicine I, Endocrinology and Metabolism, Heidelberg University, 69115 Heidelberg, Germany.
Schneider
J G
JG
Dugi
K A
KA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004622:Embryo, Mammalian; D004795:Enzyme Stability; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014162:Transfection; D014304:Triolein / Q000378:metabolism
D018076:DNA, Complementary; D014304:Triolein; D008071:Lipoprotein Lipase