Biochemical characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase
Biochemical characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase
复制标题
DOI：
10.1074/jbc.m208490200
复制
发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Fan, B;Rosen, BP
大肠杆菌COPA是一种铜离子转运的P型ATPase，具有铜抗性。COPA与[γ(-32)P]-ATP形成了一种磷酸化中间体。磷酸化被钒酸盐抑制，对KOH和羟胺敏感，与保守的Asp-523上形成的酰基磷酸一致。磷酸化需要一价阳离子，要么是铜(I)，要么是银(I)。二价阳离子铜(II)、锌(II)或钴(II)不能替代，表明该转运铜的P型ATPase底物是铜(I)而不是铜(II)。从十二烷基麦芽糖苷增溶的膜中提纯的CoPA同样表现出铜(I)/银(I)刺激的ATPase活性，其K-m为0.5 mM。COPA有两个N-末端的Cys(X)(2)Cys序列，Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17)和Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113)，以及在膜跨区第6段有Cys(479)-Pro-Cys(481)基序。通过突变和缺失对这些半胱氨酸残基的需求进行了研究。N-末端半胱氨酸取代或第一个Cys-(X)(2)-Cys基序缺失的突变体形成酰基磷酸中间体。从磷酸酶形成对铜的依赖性来看，突变体对铜的亲和力似乎是野生型COPA的2-3倍。相反，Cys(479)或Cys(481)中的替换导致铜抗性、转运和磷酸酶形成的丧失。这些结果表明，Cys-Pro-Cys基序的半胱氨酸残基(而不是N端的半胱氨酸残基)是COPA发挥作用所必需的。
Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma(-32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K-m for ATP of 0.5 mM. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.