The self-association of rabbit muscle phosphofructokinase (PFK) was monitored as a function of temperature, pH, and ionic strength in order to understand the thermodynamics of this aggregation process. Thermodynamic parameters obtained from the temperature study show that the dimerization of PFK is characterized by negative entropy and enthalpy changes of -270 = 5 eu and -87 ± 1 kcal/mol, respectively, with no observable change in heat capacity. This is in contrast to the formation of the tetramer, which is governed by positive entropy and enthalpy changes and a positive heat capacity change of 5000 ± 2000 cal/mol. Low ionic strength also favors the formation of the dimer without a significant influence on the tetramerization, which is enhanced by increasing the pH from 6.00 to 8.55. Furthermore, Wyman linkage analysis [Wyman, J. (1964) Adv. Protein Chem. 19, 224–285] reveals that the formation of the tetramer from the monomer between pH 6.00 and pH 8.55 involves the loss of 3.3 protons. Further analysis shows that ionization of residues with an apparent pKa of 6.9 is linked to the formation of PFK tetramers. The conclusion of this study indicates that the major noncovalent forces governing the formation of the dimer are different from those for the association of the tetramer.
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