Fructose diphosphatase from rabbit muscle: III. Isolation of enzyme-substrate and enzyme-adenosine monophosphate complexes

Abstract

The binding of substrate (fructose diphosphate) and allosteric effector (AMP) to purified rabbit muscle fructose diphosphatase has been measured at pH 7.5 and at pH 9.3. The enzyme contains approximately four binding sites for each of these ligands. The saturation curve for the formation of the enzyme-substrate complex at pH 7.5 is sigmoidal, suggesting a cooperative interaction between binding sites. The values of the microscopic association constants at this pH range from 3.0 × 10 5 m −1 to 3.5 × 10 6 m −1. The muscle enzyme has a lower affinity for the substrate than does the liver enzyme. The binding of AMP to muscle fructose diphosphatase at neutral pH is not affected by substrate or Mg 2+. The saturation curve at pH 7.5 is hyperbolic and the association constant for the enzyme-AMP complex is 1.2 × 10 6 m −1. In the presence of Mn 2+, however, there is evidence for a positive interaction between the AMP-binding sites. The affinity of the muscle enzyme for AMP is approximately 20 times greater than that of the liver enzyme.