Reytor González Mey Ling, Cornell-Kennon Susan, Schaefer Erik, Kuzmič Petr
BioKin Ltd., Watertown, MA, USA.
AssayQuant Technologies Inc., Marlborough, MA, USA.
Anal Biochem. 2017 Feb 1;518:16-24. doi: 10.1016/j.ab.2016.11.001. Epub 2016 Nov 4.
We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [Anal. Biochem.321, 457-461, 1983], the integrated rate equation contains the Michaelis constant (K) and the specificity number (k≡k/K) as adjustable parameters, but not the turnover number k. A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters. The newly proposed method is compared with the standard approach based on the "initial linear region" of the reaction progress curves, followed by nonlinear fit of initial rates to the hyperbolic Michaelis-Menten equation. A representative set of three chelation-enhanced fluorescence EGFR kinase substrates is used for experimental illustration. In one case, both data analysis methods (linear and nonlinear) produced identical results. However, in another test case, the standard method incorrectly reported a finite (50-70 μM) K value, whereas the more rigorous global nonlinear fit shows that the K is immeasurably high.
我们提出，遵循米氏反应机制的酶反应进程可以通过一个新推导的代数方程方便地描述，该方程包含朗伯欧米伽函数。遵循诺思罗普的观点[《分析生物化学》321, 457 - 461, 1983]，积分速率方程包含米氏常数（K）和特异性常数（k≡k/K）作为可调参数，但不包含周转数k。对常规全局拟合方法的一种改进涉及将名义底物浓度作为固定或交替优化的模型参数进行组合处理。将新提出的方法与基于反应进程曲线“初始线性区域”的标准方法进行比较，随后将初始速率对双曲线米氏方程进行非线性拟合。使用一组具有代表性的三种螯合增强荧光EGFR激酶底物进行实验说明。在一种情况下，两种数据分析方法（线性和非线性）产生了相同的结果。然而，在另一个测试案例中，标准方法错误地报告了一个有限（50 - 70 μM）的K值，而更严格的全局非线性拟合表明K值高到无法测量。
