Kinetic properties of human pancreatic carboxylesterase.
Fractionation of pancreatic juice by heparin-Sepharose and cholate-Sepharose affinity chromatography indicated that pancreatic carboxylesterase can be separated from pancreatic lipase with the former retained and the latter unretained by both columns. The chromatographic behavior of pancreatic carboxylesterase was found to be similar to that of human milk bile salt-activated lipase. The partially purified pancreatic carboxylesterase had a specific activity of 30 mumol/min per mg protein when assayed with p-nitrophenyl acetate. The reaction mechanism of human pancreatic carboxylesterase was studied using p-nitrophenyl acetate as substrate and taurocholate as activator. The reaction of the enzyme was found to follow a rapid-equilibrium random mechanism. Because of the presence of basal activity, the role of taurocholate can be considered as a non-essential activator and the dissociation constant for the enzyme-taurocholate binary complex was determined to be 0.20 mM. The activation effect of taurocholate consists in increasing the affinity of the enzyme to the substrate (5.6-fold) and in increasing the Vmax (2.3-fold). Based on the kinetic property of human pancreatic carboxylesterase and human milk bile salt-activated lipase with p-nitrophenyl acetate, cholesterol oleate and triolein as substrate, we conclude that they share common substrate specificity but show minor differences in kinetic parameters. Fluorescence studies indicated that both enzymes showed a decreased intrinsic tryptophanyl fluorescence upon incubation with taurocholate. This indicates that bile salt caused a conformational change of the enzymes, with a resultant decreased hydrophobicity in the microenvironment of tryptophan residues.
D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects*; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008049:Lipase / Q000302:isolation & purification*; D008875:Middle Aged; D008892:Milk / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism; D010189:Pancreatic Juice / Q000201:enzymology*; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D009596:Nitrophenols; C008642:4-nitrophenyl acetate; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D008049:Lipase