Enzyme kinetics determined using calorimetry: A general assay for enzyme activity?
Enzyme kinetics determined using calorimetry: A general assay for enzyme activity?
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DOI：
10.1006/abio.2001.5218
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发表时间：
2001-09-15
影响因子：
2.9
通讯作者：
Gomez, J
中科院分区：
文献类型：
--
作者：
Todd, MJ;Gomez, J
提出了两种使用等温滴定微量热法测定酶动力学常数的技术。这些方法基于反应速率和产生的热功率（热量/时间）之间的比例。 (i) 可以用增加量的底物滴定酶，同时保持伪一级条件。 (ii) 在单次注入后，可以连续监测随着基质耗尽而发生的热功率变化。两种方法都可以在单个实验中进行高精度的动力学表征，并且可用于测量酶抑制。使用每个 EC 分类中的代表性酶来证明适用性，包括 (i) DHFR 的氧化还原活性 (EC 1.5.1.3)； (ii)肌酸磷酸激酶(EC 2.7.3.2)和己糖激酶(EC 2.7.1.1)的转移酶活性； (iii)幽门螺杆菌脲酶(EC 3.5.1.5)、胰蛋白酶(EC 3.4.21.4)和HIV-1蛋白酶(EC 3.4.21.16)的水解活性； (iv) 肝素酶的裂合酶活性（EC 4.1.1.7）； (v)丙酮酸羧酸酯的连接酶活性(EC 6.4.1.1)。这种无损方法是完全通用的，可以使用生理基质对光谱不透明溶液中的反应进行精确分析。这种通用测定法可能在功能基因组学中具有广泛的适用性。 (C) 2001 年学术出版社。
Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Heliobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics. (C) 2001 Academic Press.