Grosse R, Eckert K, Malur J, Repke K R
Acta Biol Med Ger. 1978;37(1):83-96.
The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.
本文利用与NBD - 氯化物反应导致的失活作为评估酶与各种效应物结合的分析工具，描述了ATP、Na⁺和K⁺与（NaK）-ATP酶的相互作用。1. 与NBD - 氯化物反应导致（NaK）-ATP酶失活，在所有研究条件下均呈现准一级反应速率，这基于催化中心或其附近巯基的烷基化。结合在催化中心的ATP通过保护这些巯基不被烷基化，防止酶被NDD - 氯化物失活。Na⁺和K⁺要么通过影响ATP结合间接影响巯基对NBD - 氯化物的反应性，要么通过改变催化中心的构象更直接地影响。基于这些相互关系，分析了各种效应物与酶的相互作用。2. 通过我们的方法测定的各种核苷酸的K'D值与通过独立方法获得的值一致。首次表明，每个酶分子存在两个催化中心。它们对ATP和ADP表现出高亲和力或低亲和力，这显然是由于酶的半单位通过亚基间通讯（“半位点反应性”）的反协同相互作用引起的。3. 在没有ATP的情况下，（NaK）-ATP酶的Na⁺或K⁺结合对催化中心巯基的反应性产生相反的影响，反映了它们构象的不同变化。这与Na⁺和K⁺对（NaK）-ATP酶某些部分反应的众所周知的拮抗作用相对应。改变巯基反应性所需的Na⁺和K⁺浓度相当高，即，在没有酶与ATP结合的情况下，Na⁺和K⁺的离子载体中心不易用于阳离子络合。4. Na⁺对ATP与酶的结合没有影响，也不影响与NBD - 氯化物的失活反应，而K⁺通过降低ATP结合显著降低了ATP的保护作用。酶 - ATP复合物对K⁺的亲和力比游离酶高两个多数量级。K⁺结合的酶 - ATP复合物的Na⁺结合逆转了K⁺结合的作用，并产生了明显超过游离酶与ATP络合的保护作用。因此，酶分子同时携带Na⁺和K⁺的离子载体中心。5. 每个酶分子同时存在Na⁺和K⁺的离子载体中心以及具有反协同相互作用的两个催化中心这一发现，证实了（NaK）-ATP酶泵机制的翻转概念的相应基本预测，并解释了（NaK）-ATP酶转运和酶活性的一些特殊动力学特征。
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