Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT.
Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT.
复制标题
使用 DYNAFIT 定位三步水解酶催化反应的速率决定步骤。
DOI：
10.1016/j.bbapap.2008.02.004
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Sheehy,JohnPaul
中科院分区：
文献类型：
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作者：
Zhang,Daoning;Kovach,IldikoM;Sheehy,JohnPaul
在pH8.0 -8.4和25.0±0.1 °C条件下，用动态曲线法研究了人α-凝血酶、人活化蛋白C和人凝血因子Xa催化寡肽4-硝基苯胺的水解反应，用DYNAFITV计算了各反应速率常数，其内部误差基本在10%以内。一个系统的战略已经开发出适合的三步连续机制，以1800至6000个时间过程的数据点，从两个到四个独立的动力学实验轮询。还计算了酶和底物浓度。个别速率常数以及再现出版的可比条件下获得的值和Michaelis-Menten动力学参数计算这些基本速率常数也在合理的范围内公布的值。为了进行比较，积分Michaelis-Menten方程也拟合到来自12组的数据。kcat和kcat/Km值与使用DYNAFIV获得的基本速率常数计算的值在15%以内。第二和第三个连续步骤的速率常数在3-4倍内，表明两者都决定了总速率。在25.0±0.1 °C下，在pH 8.4的一系列含有递增氘分数的缓冲液中，因子Xa催化的N-α-Z-d-Arg-Gly-Arg-pNA·2 HCl的水解显示出k3对缓冲液中D含量的非常强的依赖性和k2对缓冲液中D含量的中度依赖性：分馏因子为：K1为0.49±0.03，k2为0.70 ±0.05，k3为（0.32±0.03）2。
Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-α-thrombin, human activated protein C and human factor Xa were studied at pH 8.0–8.4 and 25.0±0.1 °C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis–Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis–Menten equation was also fitted to data from twelve sets. Both the kcatand kcat/Kmvalues are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3–4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-α-Z-d-Arg-Gly-Arg-pNA·2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0±0.1 °C shows a very strong dependence of k3and a moderate dependence of k2on D content in the buffer: the fractionation factors are: 0.49±0.03 for K1,0.70±0.05 for k2, and (0.32±0.03)2for k3.