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 [human] 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 .times. 10-8 M and Km(TG) = 2.5 .times. 10-3 M. The dissociation constant (KS) of the enzyme-TG binary complex was determined from Scatchard plots to be 7.6 .times. 10-8 M. Heparin was 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 interacted with dansylated bovine milk LPL, increasing its fluorescent emission by inducing a conformational change in the enzyme. The LPL-catalyzed reaction probably 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.