Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.
The exchange of 18O 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., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-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 18O 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 kcat/Km, 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 kcat/Km 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.
Muderhwa
J M
JM
Hormel Institute, University of Minnesota, Austin 55912.
Schmid
P C
PC
Brockman
H L
HL
D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002384:Catalysis; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D010103:Oxygen Isotopes; D010179:Pancreas / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D014867:Water / Q000378:metabolism
D005227:Fatty Acids; D010103:Oxygen Isotopes; D010743:Phospholipids; D014867:Water; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase