Observation of Asymmetry amongst Nucleotide Binding Sites of F₁-ATPase of Escherichia coli by ³¹P NMR Spectroscopy

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.