Tan Ying-Cai, Wu Hao, Wang Wei-Ning, Zheng Yi, Wang Zhi-Xin
National Laboratory of Biomacromolecules, Center for Molecular Biology, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China.
Anal Biochem. 2002 Nov 15;310(2):156-62. doi: 10.1016/s0003-2697(02)00382-2.
A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).
本文提出了一种用于研究鸟嘌呤核苷酸交换因子催化反应动力学的新型分光光度法。该方法包含两个偶联酶系统：（a）。刺激小GTP酶固有GTP水解反应的GTP酶激活蛋白；（b）。嘌呤核苷酸磷酸化酶及其发色底物7-甲基-6-硫代鸟苷，用于定量生成的无机磷酸盐。连续偶联酶系统用于表征小GTP酶RhoA与其鸟嘌呤核苷酸交换因子Lbc和Dbl之间的相互作用。此处获得的动力学参数表明，这些鸟嘌呤核苷酸交换因子与RhoA相互作用的动力学机制没有显著差异。米氏常数测定为约1微摩尔，催化常数k(cat)约为0.1秒^(-1)。
