Chipman D M, Lev A
Biochemistry. 1983 Sep 13;22(19):4450-9. doi: 10.1021/bi00288a016.
Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.
用戊二醛处理钠钾 - ATP酶的电鳐膜制剂会导致该蛋白质的酶促行为发生不可逆变化，这些变化并非由于活性位点的修饰所致。当在含有钾离子且无钠离子的介质中进行戊二醛处理时，如此产生的“钾离子修饰酶”在酶学性质上呈现出以下变化：ATP的稳态磷酸化以及ATP - ADP交换速率降至对照值的约40%，而钠钾 - ATP酶活性降至对照值的约15%。磷酸酶活性降低很少，但反应的钾离子激活参数发生了变化，从对照中K0.5约等于5 mM且nH = 1.9变为钾离子修饰酶中K0.5约等于0.5 mM且nH = 1。核苷酸对磷酸酶活性抑制的表观抑制常数KI(app)显著增加。还观察到了ATP酶反应对阳离子依赖性的变化。所有这些效应都可以通过假设当蛋白质亚基处于E2构象时表面基团的交联将酶的内在构象平衡向E2方向移动来解释。我们考虑了钾离子结合与构象平衡之间耦合的最简单数学模型，其中等效的钾离子位点必须同时处于相同状态。如果假设钾离子修饰酶中磷酸酶活性的钾离子激活反映了E2对钾离子的亲和力，那么如果只有两个钾离子位点，天然酶中磷酸酶活性的行为就能得到拟合，其在E2中的亲和力比在E1中高80倍，且E2在E1平衡中的平衡常数约为250。相同的位点可以解释ATP水解过程中去磷酸化的激活。无论选择何种模型，E2形式的催化作用都必须需要钾离子，而钾离子不能仅仅是“别构激活剂”。在含有钠离子的介质中用戊二醛修饰的酶也具有有趣的性质，但其合理性解释起来不太直接。钠钾 - ATP酶活性比“部分反应”受到的抑制更严重，就像在钾离子修饰酶中一样。我们认为这是酶修饰的一个普遍预期结果。
