19F NMR Investigation of F1-ATPase of Escherichia coli Using Fluorinated Ligands

  • Jung, Seun-Ho (Department of Microbiological Engineering, College of Engineering, Konkug University) ;
  • Kim, Hyun-Won (Department of Biochemistry and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine)
  • Published : 1998.01.31

Abstract

Asymmetry amongst nucleotide binding sites of Escherichia coli $F_1$-ATPase was examined using $^{19}F$ NMR signal from fluorinated analogs of adenine nucleotides bound to nucleotide binding sites. ADP-$CF_2-{PO_3}^{2-}$ showed no inhibitory effect to $F_1$-ATPase. But ADP-CHF-${PO_3}^{2-}$ (racemic mixture) showed competitive inhibition of $F_1$-ATPase with $K_i$ of $60\;{\mu}m$. ADP-CHF-${PO_3}^{2-}$ shows only negligible binding to $EF_1$ in the absence of $Mg^2+$. With the addition of $Mg^2+$ to the medium, the $^{19}F$ resonance of free ADP-CHF-${PO_3}^{2-}$ disappeared and the new broad resonances appeared. Appearance of more than two new asymmetric resonances following the binding of ADP-CHF-${PO_3}^{2-}$ to $EF_1$ may indicate that at least one of the isomers showed split resonances. This may suggest that the region between ${\alpha}$-and ${\beta}$-phosphate of ADP-CHF-${PO_3}^{2-}$ which is bound to catalytic sites is experiencing a different environment at different sites.

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