Lowe A G, Smart J W
Biochim Biophys Acta. 1977 Apr 12;481(2):695-705. doi: 10.1016/0005-2744(77)90303-5.
The hydrolysis of [gamma-32P]ATP by porcine brain (Na+ + K+)-stimulated ATP phosphohydrolase (EC 3.6.1.3) has been studied at 28 degree C in a rapid mixing quenched-flow apparatus. An "early burst" in the release of Pi from ATP has been observed when the enzyme is mixed with ATP, Na+ and a relatively high concentration of K+ (10 mM) but the burst is less pronounced with 0.5 mM K+. This "early burst" of Pi release is suppressed when the enzyme is pre-mixed with 10 mM K+ or 20% (v/v) dimethylsulphoxide before mixing with ATP and Na+, and premixing of enzyme with Na+ antagonizes this effect of dimethylsulphoxide. The results have been analysed by a non-linear least squares regression treatment and are consistent with a mechanism involving three steps, one of which may be a relatively slow change in enzyme conformation following release of Pi from its covalent linkage with the enzyme, in addition to formation of the enzyme-substrate complex. Rate constants (and S.E.) for these steps have been calculated and the roles of phospho-enzyme and other intermediates in the reaction mechanism of the transport ATPase are dicussed.
在快速混合淬灭流动装置中，于28℃研究了猪脑（Na⁺ + K⁺）刺激的ATP磷酸水解酶（EC 3.6.1.3）对[γ-32P]ATP的水解作用。当该酶与ATP、Na⁺以及相对高浓度的K⁺（10 mM）混合时，观察到从ATP释放Pi出现“早期爆发”，但在0.5 mM K⁺时这种爆发不太明显。当酶在与ATP和Na⁺混合之前预先与10 mM K⁺或20%（v/v）二甲基亚砜预混合时，Pi释放的这种“早期爆发”受到抑制，并且酶与Na⁺的预混合会拮抗二甲基亚砜的这种作用。通过非线性最小二乘回归处理对结果进行了分析，结果与一个涉及三个步骤的机制一致，除了形成酶 - 底物复合物外，其中一个步骤可能是Pi从与酶的共价键释放后酶构象的相对缓慢变化。已经计算了这些步骤的速率常数（及标准误差），并讨论了磷酸化酶和其他中间体在转运ATP酶反应机制中的作用。
