REGULATION OF FATTY-ACID O-18 EXCHANGE CATALYZED BY PANCREATIC CARBOXYLESTER LIPASE .1. MECHANISM AND KINETIC-PROPERTIES

The exchange of O-18 between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., O-18,O-18-DA half arrow right over half arrow left O-18,O-16-DA half arrow right over half arrow left O-16,O-16-DA. This indicates that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of O-18 exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of k(cat)/K(m), 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant k(cat)/K(m) was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.