Robinson J D, Guerra M, Davis R L, Steinberg M
Nauchnye Doki Vyss Shkoly Biol Nauki. 1990(6):97-106.
To help characterize the Na,K-ATPase active site with enzyme incorporated into phospholipid vesicles, the activities with alternative substrates were compared, 22Na/Na-transport was equivalent with ATP, CTP, carbamylphosphate and acetylphosphate, but slower with CTP, 3-O-methylfluoresceinphosphate (3-O-MFP), nitrophenylphosphate and umbelliferonephosphate. It indicates a slower rate of formation of phosphorylating enzyme complex in conformation position of E1 (E1P) when the second group of substrates is bound with enzyme active center. 22Na/K-transport was half as effective with CTP as with ATP and was far slower with the other substrates. It indicates a more stringent selectivity at the low-affinity site of enzyme in conformation E2 that accelerates the slow step of this transport mode. Although enzyme modification with fluoresceinisothiocyanate blocks the high-affinity site to ATP, the K-phosphatase reaction catalyzed by E2 is retained, even with a substrate, 3-O-MFP, that binds to the adenine pocket. Dimethylsulfoxide inhibits hydrolysis of the nucleotides and of the carboxylic phosphate substrates of the K-phosphatase reaction, but stimulates hydrolysis of the phenolic phosphate substrates (nitrophenylphosphate and umbelliferone phosphate) which normally are hydrolyzed more slowly than the other substrates. On the basis of these data the authors propose the model of Na,K-ATPase active center.
为了利用掺入磷脂囊泡中的酶来表征钠钾ATP酶的活性位点，比较了其对替代底物的活性。22Na/Na转运对ATP、CTP、氨甲酰磷酸和乙酰磷酸的反应相当，但对CTP、3 - O - 甲基荧光素磷酸酯（3 - O - MFP）、硝基苯磷酸酯和伞形酮磷酸酯的反应较慢。这表明当第二组底物与酶活性中心结合时，在E1（E1P）构象位置形成磷酸化酶复合物的速率较慢。22Na/K转运对CTP的效率只有对ATP的一半，对其他底物的效率则慢得多。这表明在E2构象的酶低亲和力位点具有更严格的选择性，从而加速了这种转运模式的慢步骤。尽管用异硫氰酸荧光素修饰酶会阻断对ATP的高亲和力位点，但E2催化的K - 磷酸酶反应仍然保留，即使是与结合到腺嘌呤口袋的底物3 - O - MFP反应时也是如此。二甲基亚砜抑制核苷酸和K - 磷酸酶反应的羧酸磷酸底物的水解，但刺激酚类磷酸底物（硝基苯磷酸酯和伞形酮磷酸酯）的水解，这些底物通常比其他底物水解得更慢。基于这些数据，作者提出了钠钾ATP酶活性中心的模型。
