Brooks H B, Davidson V L
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):211-3. doi: 10.1042/bj2940211.
The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.
分析停流动力学数据最常用的方法需要进行一系列测量，其中一种反应物的浓度变化要显著大于另一种反应物的浓度。对于酶催化反应，这可能无法实现，因为酶 - 底物复合物的解离常数通常与停流研究中经常必须使用的高浓度酶处于同一数量级。本文提出了一种数据分析的替代方法，该方法可根据停流动力学数据的初始速率确定微观速率常数，其中底物浓度在与酶大致相同的范围内变化。此方法还提供了一种简单而准确的方法来确定k4，即逆向反应的速率。该方法已用于描述一种醌蛋白（甲胺脱氢酶）和一种铜蛋白（蓝铜蛋白）之间的生理电子转移反应。在20摄氏度时，从甲胺脱氢酶到蓝铜蛋白的电子转移反应速率为24 s-1，复合物形成的解离常数为1.9 microM。
