pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains.
pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains.
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ATP 与肌浆网 ATP 酶结合的 pH 和镁依赖性。
DOI：
10.1016/s0021-9258(19)40236-6
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
F. Guillain
中科院分区：
文献类型：
--
作者：
J. Lacapere;N. Bennett;Y. Dupont;F. Guillain
核苷酸结合肌浆网囊泡进行了研究，在没有钙使用过滤和荧光测量。放射性核苷酸的结合在浓度高达0.1 mM的过滤试验得到了一个ATP结合位点/肌浆网ATP酶分子的化学计量。当在钙的存在下在其他类似条件下测量时，ATP酶速度上升4-8倍（取决于pH和镁浓度）。ATP和ADP的结合增强了肌浆网ATP酶的内源荧光，过滤和荧光测量均表明，无金属ATP的结合与pH无关（Kd = 20-25 μ M），但镁的存在诱导Mg·ATP复合物结合的pH依赖性（在pH 6.0时Kd = 10 μ M，在pH 8.0时Kd = 1.5 μ M）。无金属ADP的结合是pH依赖性的，但不受镁的影响。高镁浓度抑制核苷酸结合。这些结果表明，ATP相互作用的两个不同的结构域的Ca-ATP酶，形成催化位点。第一个结构域可以结合底物的腺嘌呤部分，并且ADP结合的pH依赖性表明His 683参与该区域。催化位点的第二个结构域可以结合Mg·ATP复合物的γ-磷酸和镁离子，并构成底物和酶之间的静电相互作用的位点。
Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.