Crystallization and Properties of Phosphofructokinase from Clostridium pasteurianum

Phosphofructokinase has been purified from Clostridium pasteurianum and prepared in crystalline form in the presence and the absence of ATP. The enzyme preparation is homogeneous as shown by sedimentation velocity, sedimentation equilibrium, and acrylamide gel electrophoretic techniques. The molecular weight of the clostridial phosphofructokinase is 144,000. The enzyme is dissociated to subunits with molecular weight of about 35,000 in guanidine or urea or by treatment with maleic anhydride. Peptide mapping of a tryptic digest of the enzyme, acrylamide gel electrophoresis of the enzyme in urea, and the electrophoresis of the maleylated phosphofructokinase indicate that all these subunits are identical. Based on these observations, it is concluded that clostridial phosphofructokinase consists of 4 identical subunits. Kinetic studies show that the enzyme exhibits sigmoidal kinetics with respect to fructose 6-phosphate at all levels of ATP. ADP normalizes this initial velocity pattern to yield Michaelis-Menten kinetics. Unlike mammalian phosphofructokinases, the clostridial enzyme is not significantly inhibited by ATP, phosphoenolpyruvate, or citrate. The activity of the enzyme is absolutely dependent on NH 4 + and Mg ++ , and a possible physiological significance of the former cation is discussed.