Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Observation of Asymmetry amongst Nucleotide Binding Sites of F1-ATPase of Escherichia coli by 31P NMR Spectroscopy

  • Jun, Nam-Kung (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine) ;
  • Sohn, Joon-Hyung (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine) ;
  • Yeh, Byung-Il (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine) ;
  • Choi, Jong-Whan (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine) ;
  • Kim, Hyun-Won (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine)
  • Received : 2010.11.01
  • Accepted : 2010.12.06
  • Published : 2011.02.20
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Abstract

It was regarded that the $^{31}P$ resonances of inherent nucleotides in $F_1$-ATPase (EF1), as large as 380KDa, could not be observed by $^{31}P$ NMR spectroscopy. However, our $^{31}P$ NMR spectroscopy could differentiate between different nucleotide binding sites on EF1 from Escherichia coli. When EF1 was prepared in the absence of $Mg^{2+}$, EF1 contained only ADP. Multiple $^{31}P$ resonances from $\beta$-phosphates of ADP bound to the EF1 were observed from the enzyme prepared without $Mg^{2+}$, suggesting asymmetry or flexibility amongst nucleotide binding sites. $^{31}P$ resonances from enzyme bound ATP could be observed only from EF1, when the enzyme was prepared in the presence of $Mg^{2+}$. This $Mg^{2+}$ dependent ATP binding was very tight that, once bound, nucleotide could not be removed even after removal of $Mg^{2+}$. $^{31}P$ NMR proved to be a valuable tool for investigating phosphorous related enzymes.

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