Bernard C, Buc J, Piéroni G
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
Lipids. 1996 Mar;31(3):261-7. doi: 10.1007/BF02529872.
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.
提出了一个新的概念，用于量化脂解过程中发生的非均相催化过程中的底物浓度。评估并定义了每单位体积可吸附到脂质底物界面的蛋白质（酶）分子数量，将其作为蛋白质（酶）结合位点的体积浓度（PEBS）。以猪胰脂肪酶（EC 3.1.1.3）作为模型酶，通过测定酶对脂质底物的饱和度，在各种测定条件下测量了最大PEBS浓度。利用算盘关联每个实验条件下脂质底物浓度（M）与PEBS浓度（M），以PEBS的体积浓度来表示动力学数据。因此，由于数据以相同的单位表示，就可以对用各种酶和脂质界面获得的数据进行比较。对于胰脂肪酶，分别以三油精和三丁酰甘油为底物，得到的Km值分别为2.7和7.5 nM PEBS，并且从Scatchard图中还得到了约9 nM PEBS的KD值。此外，发现PEBS的平均表面密度为10×10(11)个分子·cm-2，这是吸附在酰基甘油 - 水界面的结构蛋白和酶通常获得的值，这一发现支持了PEBS概念代表蛋白质分子在脂质界面吸附空间的观点。
