A steady-state kinetic method for the verification of the rapid-equilibrium assumption in allosteric enzymes.
A steady-state kinetic method for the verification of the rapid-equilibrium assumption in allosteric enzymes.
复制标题
用于验证变构酶快速平衡假设的稳态动力学方法。
DOI：
10.1016/0003-2697(92)90384-j
复制
发表时间：
1992
影响因子：
2.9
通讯作者：
Reinhart,GD
中科院分区：
文献类型：
--
作者：
Symcox,MM;Reinhart,GD
本文提出了一种方法来测试快速平衡假设的有效性，因为它可能适用于变构酶，只使用稳态动力学数据。该方法是基于这样一种认识，即在底物浓度的极限和饱和条件下得到的变构配体的表观解离常数的比值，即使在一般的稳态情况下，也必须产生底物与变构配体之间耦合参数的热动力值。如果发现该值等于表观耦合参数，由底物在没有变构配体和饱和存在时得到的米切里斯常数的极限值之比确定，则可以正确地认为底物在稳态下有效地与酶实现了结合平衡。通过检测ADP对牛肉心脏线粒体nadd依赖性异柠檬酸脱氢酶的激活，证明了该方法的实用性和局限性。
A method for testing the validity of the rapid-equilibrium assumption as it might apply to allosteric enzymes using exclusively steady-state kinetic data is presented. The method is based upon a recognition that the ratio of apparent dissociation constants for the allosteric ligand, obtained under conditions of limiting and saturating substrate concentration, must yield the thermodynamic value for the coupling parameter between the substrate and allosteric ligand even in the general steady-state case. If this value is found to be equal to the apparent coupling parameter determined from the ratio of limiting values of the Michaelis constant for substrate obtained in the absence and saturating presence of the allosteric ligand, then the substrate can be correctly viewed as effectively achieving a binding equilibrium with the enzyme in the steady-state. The utility and limitations of this method are demonstrated by examining the ADP activation of beef heart mitochondrial NAD-dependent isocitrate dehydrogenase.