Wang_1981_J.Biol.Chem_256_10198

Further studies on human milk bile salt-activated lipase were performed to provide kinetic and additional chemical characterizations of this enzyme. The enzyme was homogeneous by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with an isoelectric point of 3.7. A unique feature of the amino acid composition of this enzyme was a high proline content (13 mol %). Results of carbohydrate analyses indicated that the enzyme was a glycoprotein containing fucose, galactose, glucosamine, galactosamine, and sialic acid. Kinetic studies were performed with various water-soluble esters (p-nitrophenyl acetate, 1-monoacetin, 1-monobutyrin, and 1-monocaprylin) as substrate and taurocholate as activator. In the presence of a saturating level of taurocholate, the enzyme reaction was demonstrated to follow a rapid equilibrium random uni bi mechanism. Also, these kinetic studies indicated the formation of an enzyme-activator-substrate ternary complex through a random pathway. The mechanism of the activation by taurocholate was due to its enhancement of the binding of the enzyme to the substrate (6.2-fold) and its enhancement of the rate of conversion from enzyme-substrate transitory complex to the products (1.57-fold) when examined with p-nitrophenyl acetate as substrate.