The kinetics of heparin inhibition of the esterase and basal lipase activities of lipoprotein lipase

Abstract

The kinetics of inhibition of the esterase and lipase activities of bovine milk lipoprotein lipase (LPL) were compared. The esterase LPL activity against emulsified tributyrylglycerol was not affected by the enzyme activator apolipoprotein C-II (C-II) and amounted to about 15% of the “plus activator” lipase enzyme activity. Heparin at concentrations of 20 μg/ml inhibited 25% of the esterase activity. The reaction followed Henri-Michaelis-Menten kinetics and the inhibition by heparin followed a linear, intersecting, noncompetitive kinetic model. On the other hand, the basal lipase activity of LPL against emulsified trioleoylglycerol (TG) was very sensitive to inhibition by heparin: 1 μg/ml inhibited about 80% of the reaction and 3 μg/ml drove the reaction to zero. The velocity curve for the uninhibited basal LPL activity was sigmoidal with an apparent n H(TG) of 2.94. Heparin inhibited the lipase activity competitively: heparin decreased n H(TG) and increased [TG] 0.5 6.4-fold, while TG decreased the n H(Heparin) from 2.14 to 0.95 and caused a 3-fold increase in [Heparin] 0.5. C-II, at concentrations lower than 2.5 × 10 −8 m (i.e., lower than K A ), countered the inhibitory effects of heparin: at constant inhibitor concentrations, C-II increased n H(TG) from 1.78 to 2.52 and decreased [TG] 0.5 about 10-fold; it also increased the apparent V max At the lower C-II concentrations, n H(C-II) was approximately equal to 1.0 and increasing the TG concentrations decreased [C-II] 0.5 from 3.8 × 10 −8 to 8.5 × 10 −9 m, with no effect on the n H(C-II). At the higher C-II concentrations, n H(C-II) was 2.5 and TG decreased [C-II] 0.5 about 2-fold with no effect on the n H(C-II). In the absence of heparin, C-II had no effect on n H(TG) nor on [TG] 0.5, but it increased the apparent V max. On the other hand, TG had no effect on n H(C-II) nor on [C-II] 0.5, but at any given C-II concentration, the reaction velocity increased with increasing TG concentrations. It is concluded that TG and heparin as well as C-II and heparin are mutually exclusive and that lipoprotein lipase is a multisite enzyme, possibly a tetramer, with three high-affinity catalytic sites, and an equal number of sites for C-II and heparin per oligomer. However, LPL differs from classical allosteric enzymes in that its activator has no effect on substrate cooperativity nor on [S] 0.5; its only effect is to increase V max by increasing the catalytic rate constant k p by inducing conformational changes in the enzyme.