Specificity of rabbit muscle phosphofructokinase for nucleotides as substrates or as allosteric effectors.

Various ATP and AMP analogs with modifications in the base moiety or in the polyphosphate chain were tested as substrates and/or as allosteric effectors of rabbit muscle phosphofructokinase. The significance of different structural elements for the nucleotide-enzyme interaction is discussed. While all investigated triphosphate analogs with a modified purine base are substrates for phosphofructokinase, those with a modified polyphosphate chain are competitive inhibitors. 5′-Adenylyl-(β,γ-methylene) diphosphonate, which is a weak competitive inhibitor, is shown to have a high affinity for the allosteric site of phosphofructokinase. Among the investigated monophosphate analogs only adenosine-N¹-oxide 5′-monophosphate can reverse the inhibitory effect of excessive ATP. A qualitative correlation is found between the quenching of the phospho-fructokinase-8-anilino-1-naphthalene-sulfonate fluorescence and the ability of the nucleotide analogs to act as substrates or as allosteric effectors of phosphofructokinase. It is concluded that the interaction of ATP with the allosteric site is more complex than that with the substrate site and requires both an intact adenine moiety and an intact terminal phosphate group for full activity.