Binding of ATP and 1,N6-ethenoadenosine triphosphate to rabbit muscle phosphofructokinase

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Abstract
The binding of 1,N6-ethenoadenosine triphosphate (.epsilon.ATP) to rabbit muscle phosphofructokinase was studied by fluorescence and circular dichroism, and compared with that of its counterpart, ATP. Muscle phosphofructokinase binds 11.3 .+-. 1.2 mol of .epsilon.ATP/tetramer with an average Kd of 60 .mu.M. This is in agreement with the report of 3 ATP binding sites/phosphofructokinase protomer. The binding of .epsilon.ATP is relatively homogeneous in comparison with the biphasic binding of ATP. Saturating concentrations of ATP, GTP and ADP displace about 80% of the bound .epsilon.ATP from the enzyme, whereas [fructose 1,6-diphosphate] and AMP displace only 27%. Citrate, on the other hand, enhances the affinity of phosphofructokinase for .epsilon.ATP. The effects of the binding of ATP and .epsilon.ATP on the conformation of enzyme were also compared. Binding of ATP results in increases in both the local rigidity and the ellipticity of the tryptophanyl side chains, whereas binding of .epsilon.ATP causes a slight decrease in the local rigidity and has virtually no effect on the ellipticity.