Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration |
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Authors: | Christine Bernard Jean Buc Gérard Piéroni |
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Affiliation: | (1) Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 09, France;(2) Present address: INSERM U-130, 18, Avenue Mozart, 13009 Marseille, France;(3) Present address: LCB, CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 09, France |
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Abstract: | 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,K
m values of 2.7 and 7.5 nM PEBS were obtained, respectively, andK
D values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of
PEBS was found to be 10×1011 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. |
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