Schuurmans Stekhoven F M, Swarts H G, Zhao R S, de Pont J J
Biochim Biophys Acta. 1986 Oct 9;861(2):259-66. doi: 10.1016/0005-2736(86)90428-1.
The nucleotide specificity for the E2K----E1K conformational transition in (Na+ + K+)-ATPase as the key step for overall hydrolytic activity and coupled cation transport has been investigated. Use has been made of tryptic inactivation, which is biexponential in time for the enzyme in the presence of Na+ with or without nucleotides (E1 conformation) and monoexponential in the presence of K+ (E2 conformation). ATP, AdoPP[NH]P and CTP in order of decreasing effectivity induce the biphasic tryptic inactivation pattern in the presence of K+. Their order of effectivity is inversely related to the rate constant of the second (slow) phase of inactivation. In the presence of K+ and either ITP or GTP tryptic inactivation remains monoexponential, indicating that these nucleotides cannot drive the E2K----E1K transition. Tryptic inactivation has been compared with tryptic fragmentation of the alpha-subunit (apparent mol. wt. 94 kDa) of (Na+ + K+)-ATPase. In the E1 conformation (Na+ present) a 71 kDa fragment is formed during the second phase of inactivation. In the E2 conformation (K+ present) the alpha-subunit is split to fragments of 41 and 52 kDa. In the presence of K+ and ATP, ADP, AdoPP[NH]P or CTP the 71 kDa fragment is formed in amounts which decrease in the order ATP approximately equal to ADP greater than AdoPP[NH]P greater than CTP. In the presence of K+ and AMP, ITP or GTP the 71 kDa fragment is absent and only the E2 fragments are formed. From these and literature data we arrive at a specificity order for the E2K----E1K transition of ATP greater than ADP greater than AdoPP[NH]P greater than CTP greater than ITP = GTP = AMP. The same order holds for K+ transport in the K+-K+ exchange and for overall hydrolytic activity (Na+ + K+ present) with the natural nucleoside triphosphates as substrates. This marks the E2K----E1K transition as the step in the reaction mechanism that determines nucleotide specificity for (Na+ + K+)-activated hydrolysis and coupled cation transport.
研究了（Na⁺ + K⁺）-ATP 酶中 E2K----E1K 构象转变的核苷酸特异性，该转变是整体水解活性和偶联阳离子转运的关键步骤。利用了胰蛋白酶失活，在有或无核苷酸（E1 构象）存在 Na⁺的情况下，酶的失活时间呈双指数形式，而在有 K⁺（E2 构象）存在时呈单指数形式。ATP、AdoPP[NH]P 和 CTP 按有效性递减顺序在有 K⁺存在时诱导双相胰蛋白酶失活模式。它们的有效性顺序与失活第二（慢）相的速率常数呈反比。在有 K⁺和 ITP 或 GTP 存在时，胰蛋白酶失活仍为单指数形式，表明这些核苷酸不能驱动 E2K----E1K 转变。将胰蛋白酶失活与（Na⁺ + K⁺）-ATP 酶α亚基（表观分子量 94 kDa）的胰蛋白酶片段化进行了比较。在 E1 构象（存在 Na⁺）下，在失活的第二阶段形成一个 71 kDa 的片段。在 E2 构象（存在 K⁺）下，α亚基被裂解为 41 kDa 和 52 kDa 的片段。在有 K⁺和 ATP、ADP、AdoPP[NH]P 或 CTP 存在时，71 kDa 片段的形成量按 ATP 约等于 ADP 大于 AdoPP[NH]P 大于 CTP 的顺序减少。在有 K⁺和 AMP、ITP 或 GTP 存在时，不存在 71 kDa 片段，仅形成 E2 片段。根据这些数据和文献数据，我们得出 E2K----E1K 转变的特异性顺序为 ATP 大于 ADP 大于 AdoPP[NH]P 大于 CTP 大于 ITP = GTP = AMP。对于以天然核苷三磷酸为底物的 K⁺-K⁺交换中的 K⁺转运和整体水解活性（存在 Na⁺ + K⁺），同样的顺序成立。这标志着 E2K----E1K 转变是反应机制中决定（Na⁺ + K⁺）激活水解和偶联阳离子转运的核苷酸特异性的步骤。
