Burdette R A, Quinn D M
J Biol Chem. 1986 Sep 15;261(26):12016-21.
The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.
以几摩尔百分比掺入含有 Triton X - 100 的混合胶束中的脂肪酰基（脂质）对硝基苯酯，即对硝基苯辛酸酯、对硝基苯月桂酸酯和对硝基苯棕榈酸酯，是牛乳脂蛋白脂肪酶的底物。当混合胶束的组分浓度约等于或大于临界胶束浓度时，脂蛋白脂肪酶催化酯水解的时间进程可用米氏方程的积分形式来描述。因此，对积分方程进行最小二乘法拟合可通过单次运行计算界面动力学参数 Km 和 Vmax。本文描述了用于确定界面动力学参数的计算方法，并用于确定脂蛋白脂肪酶催化的内在底物脂肪酰基特异性，这反映在 kcat/Km 和 kcat 的大小上。界面脂蛋白脂肪酶催化的结果，连同先前测定的水溶性酯对硝基苯乙酸酯和对硝基苯丁酸酯的动力学参数，表明脂蛋白脂肪酶对具有中等长度脂肪酰基链的底物（即对硝基苯丁酸酯和对硝基苯辛酸酯）具有最高特异性。在脂蛋白脂肪酶催化 Triton X - 100 胶束中所含脂质对硝基苯酯水解过程中，脂肪酸产物不会引起产物抑制。亲核试剂羟胺、肼和乙二胺对脂蛋白脂肪酶催化对硝基苯月桂酸酯水解的 Km 和 Vmax 的影响与月桂酰 - 脂蛋白脂肪酶中间体的捕获一致。当羟胺作为亲核试剂时，通过分析产物月桂基异羟肟酸酯证实了该机制。因此，脂蛋白脂肪酶催化 Triton X - 100 胶束中所含脂质对硝基苯酯的水解是通过界面酰基 - 脂蛋白脂肪酶机制进行的，该机制受酰基 - 酶中间体水解的速率限制。
