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首页> 外文期刊>Biochemistry >MERCAPTIDE FORMED BETWEEN THE RESIDUE CYS70 AND HG2+ OR CO2+ BEHAVES AS A FUNCTIONAL POSITIVELY CHARGED SIDE CHAIN OPERATIVE IN THE ARG70-]CYS MUTANT OF THE METAL-TETRACYCLINE/H+ ANTIPORTER OF ESCHERICHIA COLI
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MERCAPTIDE FORMED BETWEEN THE RESIDUE CYS70 AND HG2+ OR CO2+ BEHAVES AS A FUNCTIONAL POSITIVELY CHARGED SIDE CHAIN OPERATIVE IN THE ARG70-]CYS MUTANT OF THE METAL-TETRACYCLINE/H+ ANTIPORTER OF ESCHERICHIA COLI

机译：残留的CYS70和HG2 +或CO2 +之间形成的巯基化合物在大肠埃希氏菌的金属-四环素/ H +拮抗剂的ARG70-] CYS突变体中作为功能性正电荷侧链起作用

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The bacterial tetracycline/H+ antiporter (TetA) mediates active efflux of a chelation complex of tetracycline with a divalent cation such as Mg2+, Co2+, or Mn2+ [Yamaguchi, A., Udagawa, T., & Sawai, T. (1990a) J. Biol. Chem. 265, 4809-4813]. The positive charge of Arg70 in the antiporter is important for the transport function [Yamaguchi, A., Someya, Y., & Sawai, T. (1992c) J. Biol. Chem. 267, 19155-19162]. Out of six site-directed mutants of Arg70, only the Lys70 mutant retained moderate transport activity, whereas the Ser70, Ala70, Trp70, Leu70, and Asp70 mutants had no or extremely low transport activity. In this study, we constructed the Cys70 mutant and found that the Cys70 mutant showed, unexpectedly, a significant activity comparable to that of the Lys70 mutant in the presence of Co2+ ions, whereas it showed very low activity as well as the Ala70 mutant in the presence of Mg2+ or Mn2+ ions. Hg2+, which is known to be a cysteine specific modifier but has no ability to form a complex with tetracycline, caused a dramatic increase in the V-max value of Co2+-dependent tetracycline transport mediated by the Cys70 mutant without affecting the K-m value, whereas activities of the wild-type and the Lys70 and Ala70 mutants were not affected by Hg2+. Hg2+ alone without Co2+ could not support the transport activity at all, because Hg2+ does not form a chelation complex with tetracycline. These observations suggest that a mercaptide formed between the SH group of Cys70 and Hg2+ or Co2+ works as a positively charged side chain like that of Arg or Lys. When the SH group of the Cys70 mutant was masked with modification by sulfhydryl reagents, the residual activity was no longer affected by Hg2+. Inversely, when the Cys70 mutant was preincubated with Hg2+, it was protected from the inactivation by sulfhydryl reagents. These observations also confirm the mercaptide formation between the Cys70 and a divalent cation as a functional side chain.
机译：细菌四环素/ H +反向转运蛋白（TetA）介导四环素与二价阳离子（例如Mg2 +，Co2 +或Mn2 +）的螯合复合物的主动流出[Yamaguchi，A.，Udagawa，T.，＆Sawai，T.（1990a）J生物学化学265，4809-4813]。反转运蛋白中Arg70的正电荷对于转运功能很重要[Yamaguchi，A.，Someya，Y.，＆Sawai，T.（1992c）J.化学267，19155-19162]。在Arg70的6个定点突变体中，只有Lys70突变体保留了中等的转运活性，而Ser70，Ala70，Trp70，Leu70和Asp70突变体则没有或具有极低的转运活性。在这项研究中，我们构建了Cys70突变体，发现在存在Co2 +离子的情况下，Cys70突变体意外地显示出与Lys70突变体相当的显着活性，而在Cys70突变体中却显示出非常低的活性以及Ala70突变体。 Mg2 +或Mn2 +离子的存在。 Hg2 +是已知的半胱氨酸特异性修饰剂，但不能与四环素形成复合物，导致Cys70突变体介导的Co2 +依赖性四环素转运的V-max值急剧增加，而不会影响Km值，而Hg2 +不会影响野生型以及Lys70和Ala70突变体的活性。单独的不含Co2 +的Hg2 +根本无法支持运输活性，因为Hg2 +不会与四环素形成螯合复合物。这些观察结果表明，Cys70的SH基团与Hg2 +或Co2 +之间形成的巯基像Arg或Lys一样具有带正电荷的侧链。当Cys70突变体的SH基团被巯基试剂修饰掩盖时，残留活性不再受Hg2 +的影响。相反，当将Cys70突变体与Hg2 +预孵育时，可以通过巯基试剂保护其免受灭活作用。这些观察结果也证实了Cys70和作为功能性侧链的二价阳离子之间的巯基形成。

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