title: Kinetics of bovine milk lipoprotein lipase and the mechanism of enzyme activation by apolipoprotein C-II.
abstract: The kinetics of bovine milk lipoprotein lipase (LPL) were studied in order to determine the reaction mechanism of this enzyme. Reaction velocities were determined at varying concentrations of emulsified trioleoylglycerol (TG) and different fixed concentrations of apolipoprotein C-II (C-II) or at varying C-II concentrations and different fixed concentrations of TG. Neither the apparent Km(TG) nor the apparent Km(C-II) was affected by varying the concentrations of C-II or TG, respectively. However, C-II increased the apparent Vmax for the enzyme about 20-fold. The following kinetic parameters were calculated from Lineweaver-Burk plots: Km(C-II) = 2.5 X 10(-8) M and Km (TG) = 2.5 X 10(-3) M. The dissociation constant (KS) of the enzyme-TG binary complex was determined from Scatchard plots to be 7.6 X 10(-8) M. Heparin was found to be a competitive dead-end inhibitor against both TG and C-II. Tricapryloylglycerol represented a competitive inhibitor against TG but a noncompetitive inhibitor against C-II. C-II was shown to interact with dansylated bovine milk LPL, increasing its fluorescent emission by inducing a conformational change in the enzyme. Based on these studies, it was concluded that the LPL-catalyzed reaction follows a random, bireactant, rapid-equilibrium mechanism and the role of C-II in the activation process involves an increase in the catalytic rate constant (Kp) resulting from conformational changes of LPL induced by C-II.
mesh_terms: D000818:Animals; D053304:Apolipoprotein C-II; D001053:Apolipoproteins / Q000097:blood* / Q000494:pharmacology; D001056:Apolipoproteins C; D002417:Cattle; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008433:Mathematics; D008892:Milk / Q000201:enzymology*; D011485:Protein Binding
chemical_list: D053304:Apolipoprotein C-II; D001053:Apolipoproteins; D001056:Apolipoproteins C; D008071:Lipoprotein Lipase