Yang Ying, Mandal Atin K, Bredeston Luis M, González-Flecha F Luis, Argüello José M
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Rd., Worcester, MA 01609, USA.
Biochim Biophys Acta. 2007 Mar;1768(3):495-501. doi: 10.1016/j.bbamem.2006.09.013. Epub 2006 Sep 23.
CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) across the cell membrane. Millimolar concentration of Cys dramatically increases ( congruent with 800%) the activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal ( congruent with 1 microM) together with the low Cu(+)-Cys K(D) (<10(-10)M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu(+) chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu(+), suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K(m) suggesting no changes in the E(1)<-->E(2) equilibrium. Characterization of Cu(+) transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu(+) can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu(+)-Cys complex.
CopA是一种来自嗜热古菌富铁嗜热栖热菌的ATP酶，驱动Cu(+)跨细胞膜向外移动。毫摩尔浓度的半胱氨酸可显著提高（约800%）CopA及其他P(IB)型ATP酶（大肠杆菌ZntA和拟南芥HMA2）的活性。CopA对金属的高亲和力（约1 microM）以及低的Cu(+)-半胱氨酸解离常数（<10(-10)M）表明半胱氨酸与CopA存在多方面的相互作用，可能替代了体内存在的Cu(+)伴侣蛋白。为了解释氨基酸的激活作用并进一步理解金属传递至转运ATP酶的机制，研究了半胱氨酸对CopA周转和部分反应的影响。2 - 20 mM半胱氨酸加速酶的周转，对CopA对Cu(+)的亲和力影响很小，表明是一种不依赖金属的激活。此外，半胱氨酸激活CopA的对硝基苯磷酸酶活性，尽管该活性不依赖金属。半胱氨酸加速酶的磷酸化和正向去磷酸化速率，产生更高的稳态磷酸酶水平。更快的去磷酸化可以解释在半胱氨酸存在下更高的酶周转。该氨基酸对低亲和力ATP的米氏常数没有显著影响，表明E(1)<-->E(2)平衡没有变化。对Cu(+)转运至密封囊泡的特性分析表明，半胱氨酸作用于酶的细胞质侧。然而，缺乏N端金属结合结构域（N-MBD）的截短CopA的半胱氨酸激活表明，半胱氨酸的激活不依赖于调节性N-MBD。这些结果表明半胱氨酸是CopA的非必需激活剂，在酶处于E1形式时与酶的细胞质侧相互作用。有趣的是，这些效应还指出，Cu(+)可以以游离金属或Cu(+)-半胱氨酸复合物的形式到达跨膜转运位点进入通道的细胞质开口处。
