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首页> 外文期刊>Biochemistry >Reaction Cycle of Thermotoga maritima Copper ATPase and Conformational Characterization of Catalytically Deficient Mutants
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Reaction Cycle of Thermotoga maritima Copper ATPase and Conformational Characterization of Catalytically Deficient Mutants

机译：滨海嗜热球菌铜ATP酶的反应周期和催化缺陷突变体的构象表征

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摘要

Copper transport ATPases sustain important roles in homeostasis of heavy metals and delivery ofncopper to metalloenzymes. The copper transport ATPase from Thermotoga maritima (CopA) provides anuseful system for mechanistic studies, due to its heterologous expression and stability. Its sequence comprisesn726 amino acids, including the N-terminal metal binding domain (NMBD), three catalytic domainsn(A, N, and P), and a copper transport domain formed by eight helices, including the transmembrane metalnbinding site (TMBS). We performed functional characterization and conformational analysis by proteolyticndigestion of WT and mutated (NMBD deletion or mutation) T. maritima CopA, comparing it withnArchaeoglobus fulgidus CopA and Ca2+ ATPase. A specific feature of T. maritima CopA is ATP utilizationnin the absence of copper, to forma low-turnover phosphoenzyme intermediate,with a conformation similar tonthat obtained by phosphorylation with Pi or phosphate analogues. On the other hand, formation of annactivated state requires copper binding to both NMBD and TMBS, with consequent conformational changesninvolving the NMBD and A domain. Proteolytic digestion analysis demonstrates A domain movementsnsimilar to those of other P-type ATPases to place the conserved TGES motif in the optimal position forncatalytic assistance.We also studied anH479Qmutation (analogous to one of human copper ATPase ATP7Bnin Wilson disease) that inhibits ATPase activity. We found that, in spite of the H479Q mutation within thennucleotide binding domain, the mutant still binds ATP, yielding a phosphorylation transition statenconformation. However, covalent phosphoryl transfer is not completed, and no catalytic turnover isnobserved.
机译：铜转运ATP酶在重金属的稳态和铜向金属酶的传递中起着重要作用。由于其异源表达和稳定性，来自海栖热袍菌（CopA）的铜转运ATP酶为机理研究提供了有用的系统。它的序列包含n726个氨基酸，包括N端金属结合结构域（NMBD），三个催化结构域n（A，N和P）和由八个螺旋形成的铜转运结构域，包括跨膜金属结合位点（TMBS）。我们通过WT的蛋白水解消化和突变的（NMBD缺失或突变）滨海丁酸杆菌CopA进行了功能表征和构象分析，并将其与古生化球菌CopA和Ca2 + ATPase进行了比较。海上隐孢子虫CopA的一个特殊特征是在不存在铜的情况下利用ATP来形成低周转率的磷酸酶中间体，其构象类似于通过用Pi或磷酸盐类似物进行磷酸化获得的构象。另一方面，非活化态的形成需要铜与NMBD和TMBS结合，因此构象变化涉及NMBD和A结构域。蛋白水解消化分析表明，与其他P型ATP酶的结构域运动相似，保守的TGES基序处于最佳催化辅助位置。我们还研究了H479Qmutation（类似于人类铜ATPase ATP7Bn Wilson病之一），它抑制了ATPase的活性。我们发现，尽管在核苷酸结合结构域内存在H479Q突变，该突变体仍然结合ATP，产生磷酸化过渡态构象。但是，共价磷酰基转移尚未完成，并且没有发现催化转化。

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  • 来源
    《Biochemistry》 |2009年第22期|p.4871-4880|共10页
  • 作者

    Yuta Hatori David Lewis Chikashi Toyoshima and Giuseppe Inesi;

  • 作者单位

    California Pacific Medical Center Research Institute, San Francisco, California 94107, and Institute of Molecular andCellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan;

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