Yohda M, Yoshida M
Department of Chemical Engineering, University of Tokyo.
J Biochem. 1987 Oct;102(4):875-83. doi: 10.1093/oxfordjournals.jbchem.a122128.
Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-32P]ATP and cold chase promotion were observed. However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.
通过将嗜热细菌PS3的F1 - ATP酶（TF1）与亚摩尔量的放射性ATP一起孵育，研究了其单位点催化作用。TF1在23摄氏度下的单位点催化情况与牛心线粒体F1 - ATP酶（MF1）不同。酶上的ATP水解以及产物释放很快，随后添加非放射性ATP（冷追踪）并不能促进放射性ATP的水解，这表明限速步骤不是产物释放步骤，而是ATP与酶结合的步骤。因此，未观察到所谓单位点催化的特征。在60摄氏度下，无论有无磷酸根离子，都观察到少量结合的[α,γ - 32P]ATP以及冷追踪促进现象。然而，由于通过离心凝胶过滤未检测到结合的32P1，所以TF1是否具有典型的单位点特征尚不确定。基于单位点催化的水解周转率以及稳态催化动力学分析，有人提出即使在ATP浓度低于约20微摩尔的稳态催化中，单位点催化也是占主导的。
