Abstract
The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.
Highlights
The interaction ofAMP and fructose 2,6-bisphos- It is well established that the coordinated regulation phatewithrabbitliver fructose-l,6-bisphosphatase of fructose-l,6-bisphosphataseand phosphofructokinase is has been investigated by proton nuclear magneticres- controlled byAMP and fructose 2,6-bisphosphate (1-3)
The presence of an exogenous divalent metal ion such as Binding of fructose 6-phosphateand inorganicphos- Mn2+,Zn2+,M P, or Co2+is an absolute requirement for the phate, or the regulatory inhibitor, fructose 2,6-bis- mammalian liver enzymes.Fructose-1,6-bisphosphatasefrom phosphate, results ina decrease inthe dissociation rate rabbit liver,a tetramerof identical subunits, bindsfour “strucconstant for AMP from fructose- 1,6-bisphosphatase, tural” metal ions in the absence of substrates and four addias indicated by the sharpenedAMP signals
A temper- tional metal ions in the presence of substrates at a so-called ature dependence experiment indicates that the AMP catalytic metal site (6)
Summary
Tions used in NMR experiments were Chelex-treated. NMR tubes In a recent report from this laboratory (8), we observed,. PPM denatured enzyme, gave a narrow line width (
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