Kinetics of the two‐step hydrolysis of triacylglycerol by pancreatic lipases

Abstract

Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a sequential manner. First, triacylglycerol is hydrolyzed to 1,2-diacylglycerol, which is subsequently converted to 2-monoacylglycerol. We studied the kinetics of trioleoylglycerol hydrolysis by rabbit and human pancreatic lipases. The products (acylglycerols and fatty acid) were analyzed by extraction from the reaction mixture, separation by thinlayer chromatography, and quantification by capillary gas chromatography. The first-order rate constants of trioleoylglycerol and dioleoylglycerol hydrolysis were calculated showing that both enzymes hydrolyze dioleoylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lipase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis to a higher degree than trioleoylglycerol hydrolysis. Colipase increased both rate constants similarly at high deoxycholate concentrations (35 mM), while at low concentrations (5 mM) a selectivity toward trioleoylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energies of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ . mol-' and 53.5 kJ . mol-', respectively. Upon storage, both rabbit and human pancreatic lipases showed a greater loss of activity toward dioleoylglycerol as compared to trioleoylglycerol, suggesting that different conformational elements of the enzyme molecule are responsible for the interaction with each substrate.Keywords. Pancreatic lipase ; kinetics ; rate constants ; triacylglycerol ; diacylglycerol.Pancreatic lipases catalyze the hydrolysis of triacylglycerol producing 1,2-diacyIglycerol, 2-monoacylglycerol, and fatty acids. Lipases are active at the oil/water interface in heterogeneous reaction systems (Brockman, 1984).One of the main characteristics of lipolysis by pancreatic lipases is that it takes place in a sequential manner. First, a molecule of triacylglycerol is hydrolyzed yielding one molecule of fatty acid and one molecule of 1,2-diacylglycerol, which is subsequently hydrolyzed to 2-monoacylglycerol and fatty acid. Therefore, 1,2-diacylglycerol constitutes both the initial product of lipolysis and the substrate for the second reaction. Using porcine pancreatic lipase, Constantin et al. (1960) reported a transient accumulation of 1 ,2-diacylglycerol and a late production of glycerol resulting from the cleavage of 1-monoacylglycerol after isomerization of 2-monoacylglycerol.A matter of great interest for understanding the whole mechanism of lipolysis, is the kinetics of the hydrolysis of diacylglycerol subsequent to the degradation of triacylglycerol. Despite intense research in the general field of lipases in recent years, the kinetics of this reaction remain unclear. Using trioctanoylglycerol and 1,2-dioctanoylglycero1 monolayers, Lagocki et al.(1 973) reported that the hydrolysis of the diester to 2-monooctanoylglycerol by porcine pancreatic lipase was slower than hydrolysis of the triester. The latter study was made in the abs...

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