KINETICS OF THE 2-STEP HYDROLYSIS OF TRIACYLGLYCEROL BY PANCREATIC LIPASES

Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a seq uential manner. First, triacylglycerol is hydrolyzed to 1,2-diacylglyc erol, which is subsequently converted to 2-monoacylglycerol. We studie d the kinetics of trioleoylglycerol hydrolysis by rabbit and human pan creatic lipases. The products (acylglycerols and fatty acid) were anal yzed by extraction from the reaction mixture, separation by thin-layer chromatography, and quantification by capillary gas chromatography. T he first-order rate constants of trioleoylglycerol and dioleoylglycero l hydrolysis were calculated showing that both enzymes hydrolyze diole oylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lip ase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis t o a higher degree than trioleoylglycerol hydrolysis. Colipase increase d both rate constants similarly at high deoxycholate concentrations (3 5 mM), while at low concentrations (5 mM) a selectivity toward trioleo ylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energie s of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ . mol(-1) and 53.5 kJ . mol(-1), respectively. Upon storage, both rabbi t and human pancreatic Lipases showed a greater loss of activity towar d dioleoylglycerol as compared to trioleoylglycerol, suggesting that d ifferent conformational elements of the enzyme molecule are responsibl e for the interaction with each substrate.