Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis
Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis
复制标题
通过毛细管电泳/动态前沿分析对 β-D-半乳糖苷酶酶促反应中的底物竞争进行动力学分析
DOI：
10.1016/j.jpba.2020.113390
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发表时间：
2020
影响因子：
3.4
通讯作者：
Takayanagi Toshio
中科院分区：
文献类型：
--
作者：
Mine Masanori;Mizuguchi Hitoshi;Takayanagi Toshio
采用毛细管电泳/动态前沿分析（CE/DFA）研究了酶与两种底物之间的竞争性抑制作用。以β-D-半乳糖苷酶水解邻-硝基苯基β-D-半乳糖苷和对硝基苯基β-D-半乳糖苷为模型竞争反应。将含有两种底物的样品溶液注入填充有含有酶的分离缓冲液的毛细管中。在电泳过程中发生酶水解，邻硝基苯酚和对硝基苯酚的产物不断生成并从样品区中分离出来。根据硝基苯酚和对硝基苯酚有效电泳迁移率的差异，用两种底物溶液检测到两步平台信号。米氏常数和抑制常数与平台高度。本研究证明了CE/DFA在竞争抑制分析中的准确性。
Competitive inhibition between two substrates with an enzyme is investigated by capillary electrophoresis/dynamic frontal analysis (CE/DFA). Enzymatic hydrolyses ofo-nitrophenylβ-D-galactopyranoside andp-nitrophenylβ-D-galactopyranoside withβ-D-galactosidase were examined as a model competitive reaction. A sample solution containing the two substrates was injected into a capillary filled with a separation buffer containing an enzyme. Enzymatic hydrolysis occurred during the electrophoresis, and the products ofo-nitrophenol andp-nitrophenol were continuously formed and resolved from the sample zone. Two-steps plateau signal was detected with the two-substrate solutions based on the difference in the effective electrophoretic mobility ofo-nitrophenol andp-nitrophenol. Michaelis-Menten constants and inhibition constants were determined with the plateau heights. Usefulness of CE/DFA on competitive inhibition analysis is demonstrated in this study.