Argüello J M, Kaplan J H
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
Biochemistry. 1990 Jun 19;29(24):5775-82. doi: 10.1021/bi00476a019.
Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.
用N-乙酰咪唑（NAI）处理肾钠钾ATP酶会导致钠钾ATP酶活性丧失。失活动力学可用一个模型来描述，即两类位点被NAI乙酰化。I类位点反应迅速，ATP的存在可阻止乙酰化（K0.5约为8 microM），用羟胺孵育可使失活逆转。这些数据表明I类位点是ATP结合位点处的酪氨酸残基。第二类位点反应较慢，不受ATP保护，也不能通过羟胺处理逆转。这些可能是蛋白质其他部位的赖氨酸残基。相关的钾刺激对硝基苯磷酸酶活性仅通过II类位点的乙酰化而失活；因此，与ATP结合到催化中心相关的酪氨酸残基对磷酸酶活性不是必需的。失活的酶不再具有与催化位点相关的高亲和力ATP结合，尽管低亲和力ATP效应（磷酸酶抑制和铷去封闭）仍然存在。失活的酶仍可被磷酸根磷酸化，封闭铷离子，并在酶的E1钠和E2钾形式之间经历主要构象转变。因此，NAI对钠钾ATP酶的乙酰化抑制了高亲和力ATP与催化中心的结合并导致失活。
