Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration.
A new concept is proposed for quantifying the substrate concentration during heterogeneous catalysis of the kind which occurs during lipolysis. The number of molecules of protein (enzyme) adsorbable to the lipid substrate interface per unit of volume was evaluated and defined as a volumetric concentration of protein (enzyme) binding site (PEBS). Using porcine pancreatic lipase (EC 3.1.1.3) as a model enzyme, the maximal PEBS concentration was measured under various assay conditions by determining the saturation of the lipid substrate with the enzyme. Abacuses correlating the lipid substrate concentration (M) with the PEBS concentration (M) under each experimental conditions were used to express the kinetic data in terms of a volumetric concentration of PEBS. Comparisons could thus be made between data obtained with various enzymes and lipid interfaces because they were expressed with the same unit. In the case of pancreatic lipase, using triolein and tributyrylglycerol as substrates, Km values of 2.7 and 7.5 nM PEBS were obtained, respectively, and KD values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of PEBS was found to be 10 x 10(11) molecules.cm-2, which is a value commonly obtained with structural proteins and enzymes adsorbed to an acylglyceride-water interface, this finding supports the idea that the PEBS concept represents the room in which the protein molecule adsorbs at the lipidic interface.
Bernard
C
C
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
Buc
J
J
Piéroni
G
G
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008049:Lipase / Q000378:metabolism; D050356:Lipid Metabolism; D008066:Lipolysis; D008956:Models, Chemical; D010179:Pancreas / Q000201:enzymology; D012995:Solubility; D013499:Surface Properties; D013552:Swine / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism
D014280:Triglycerides; D008049:Lipase; C005830:tributyrin