Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters.
Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters.
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DOI：
10.1016/s0021-9258(18)67195-9
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发表时间：
1986-09
期刊：
影响因子：
--
通讯作者：
R. Burdette;D. Quinn
中科院分区：
文献类型：
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作者：
R. Burdette;D. Quinn
脂肪酰基（脂质）对硝基苯基酯对硝基苯基辛酸酯、对硝基苯基月子酸酯和对硝基苯基棕榈酸酯以几摩尔%掺入与Triton X-100混合胶束中，是牛乳脂蛋白脂肪酶的底物。当混合胶束组分的浓度近似等于或大于临界胶束浓度时，脂蛋白脂酶催化酯水解的时间过程由Michaelis-Menten方程的积分形式描述。因此，对积分方程的最小二乘拟合允许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，以及先前确定的水溶性酯对乙酸对硝基苯酯和对丁酸对硝基苯酯的动力学参数，表明脂蛋白脂肪酶对具有中等长度脂肪酰基链的底物（即对丁酸对硝基苯酯和对辛酸对硝基苯酯）具有最高的特异性。脂肪酸产物在脂蛋白脂肪酶催化的Triton X-100胶束中脂质对硝基苯酯的水解过程中不会引起产物抑制。亲核试剂羟胺、肼和乙二胺对脂蛋白脂肪酶催化对硝基苯基月桂酸酯水解Km和Vmax的影响与捕获月桂基脂蛋白脂肪酶中间体一致。当羟胺为亲核试剂时，对产物十二烷基羟酸酯的分析证实了这一机理。因此，脂蛋白脂肪酶催化Triton X-100胶束中脂质对硝基苯基酯的水解通过界面酰基-脂蛋白脂肪酶机制发生，该机制受酰基-酶中间体水解的速率限制。
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.