California Pacific Medical Center Research Institute, San Francisco, California 94107, USA.
J Biol Chem. 2012 Sep 21;287(39):32717-27. doi: 10.1074/jbc.M112.373472. Epub 2012 Aug 1.
Ca(2+) (sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA)) and Cu(+) (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca(2+), demonstrated by the addition of ATP and Ca(2+) to SERCA deprived of Ca(2+) (E2) as compared with ATP to Ca(2+)-activated enzyme (E1·2Ca(2+)). Activation by Ca(2+) is slower at low pH (2H(+)·E2 to E1·2Ca(2+)) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca(2+) translocation. A "H(+)-gated pathway," demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca(2+) release by H(+)/Ca(2+) exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu(+)/H(+) exchange. As opposed to SERCA after Ca(2+) chelation, ATP7A/B does not undergo reverse phosphorylation with P(i) after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.
钙离子（肌浆内质网钙离子 ATP 酶（SERCA））和铜离子（ATP7A/B）ATP 酶通过形成磷酸酶中间物（E-P）利用 ATP，其中磷酸化势能影响结合阳离子的亲和力和取向。通过向 SERCA 中添加 ATP 和 Ca(2+)，证明了 Ca(2+)对酶的激活作用可限制 E-P 的形成，与向 Ca(2+)-激活的酶（E1·2Ca(2+)）中添加 ATP 相比，这种作用可限制 SERCA 中 Ca(2+)的耗尽（E2）。在低 pH（2H(+)·E2 到 E1·2Ca(2+)）下，Ca(2+)的激活作用较慢，并且对温度依赖性激活能的敏感性较低。另一方面，随后（正向或反向）磷酸酶处理对激活能敏感，这可缓解限制 Ca(2+)转运的构象约束。通过对 pH 变化、电荷转移和 Glu-309 突变的实验证明了一种“H(+)-门控途径”，该途径允许通过 H(+)/Ca(2+)交换释放腔内腔钙。与 SERCA 相比，ATP7A/B 对 ATP 的初始利用速度要慢得多，并且对温度依赖性激活能非常敏感，这表明其头部结构域的构象约束。与 SERCA 不同，ATP7B 磷酸酶裂解的温度依赖性比 EP 形成低得多。在 ATP7A 和 -B 中观察到 ATP 依赖性电荷转移，pH 变化时净电荷没有变化，没有证据表明存在 Cu(+)/H(+)交换。与 Ca(2+)螯合后 SERCA 不同，除非删除较大的 N-金属结合延伸片段，否则在铜螯合后，ATP7A/B 不会与 P(i)发生反向磷酸化。这归因于缺乏铜的 N-金属结合延伸与头部结构域的失活相互作用。我们得出的结论是，除了共同的（P 型）磷酸酶中间物形成外，SERCA 和 ATP7A/B 还具有独特的催化和运输机制特征。
