Lipid hydrolyses catalyzed by pancreatic cholesterol esterase. Regulation by substrate and product phase distribution and packing density.

The role of oleic acid in the regulation of the hydrolysis of cholesteryl oleate in lipid films at the air--buffer interface was investigated by using initial rate techniques. A small quantity of enzyme is rapidly adsorbed to substrate-containing films; however, a much greater, although slower, adsorption occurs if oleic acid is present. The rate constant for the slow adsorption is independent of the phase distribution of cholesteryl oleate but is markedly dependent upon both the concentration of oleic acid head groups and the acyl chain packing density in the film. Adsorption is controlled by two ionizable groups, one of which may be the carboxyl group of oleic acid. In contrast to adsorption, catalysis by the surface excess of enzyme is pH independent between 5.5 and 7.5 and is relatively specific for substrate in the monolayer phase. The second-order rate constants for the hydrolysis of cholesteryl oleate in the monolayer phase and the interfacial layer of the double-layer phase are 27 and 2 cm2 s-1 fmol-1. These results indicate that adsorption and catalysis occur at functionally. if not physically, distinct sites on the protein. The adsorption of enzyme to a hydrolysis product, oleic acid, constitutes a form of product activation which presumably helps keep it at the interface during intraluminal fat digestion. The catalytic properties of the adsorbed enzyme suggest that substrate specificities determined for cholesterol esterase in complex reaction systems may largely reflect the availability of substrate in the appropriate physical state at the lipid-water interface.