Plastid and cytosolic phosphofructokinases from the developing endosperm of ricinus communis: II. Comparison of the kinetic and regulatory properties of the isoenzymes

A steady-state kinetic analysis of plastid phosphofructokinase at pH 8.2 is consistent with the enzyme having a sequential reaction mechanism. Cytosolic phosphofructokinase probably has a similar mechanism. At pH 7.0 plastid phosphofructokinase shows cooperative binding of fructose 6-phosphate and is inhibited by higher concentrations of ATP. In contrast cytosolic phosphofructokinase shows normal kinetics at both pH 8.2 and 7.0 with respect to fructose 6-phosphate and is not inhibited by ATP. In the case of plastid phosphofructokinase the affinity for fructose 6-phosphate increases as the pH is raised from 7 to 8.2 whereas cytosolic phosphofructokinase is affected in an opposite manner. Phosphate is the principal activator of plastid phosphofructokinase since the cooperative kinetics toward fructose 6-phosphate are shifted toward Michaelis-Menten kinetics by 1 mm sodium phosphate and this concentration of phosphate relieves the inhibition by ATP. Both isoenzymes are inhibited by phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate at pH 7.2. Plastid phosphofructokinase is most strongly inhibited by phosphoenol pyruvate with the I_0.5 value varying from 0.08 to 0.5 μm depending on substrate concentrations; phosphate reverses this inhibition. In contrast cytosolic phosphofructokinase is much less inhibited by phosphoenolpyruvate with an I_0.5 approximately 1000-fold higher. Cytosolic phosphofructokinase is powerfully inhibited by 3-phosphoglycerate with an I_0.5 value of 60 μm and this appears to be the principal regulator of this isoenzyme. The two isoenzymes of phosphofructokinase in the endosperm appear, therefore, to be regulated differently. Plastid phosphofructokinase is inhibited by phosphoenolpyruvate and ATP and is activated by phosphate; whereas the cytosolic enzyme is inhibited principally by 3-phosphoglycerate and this inhibition is only partially relieved by phosphate. Some of the differences reported previously for phosphofructokinases from different plant tissues may, therefore, be due to varying ratios of the cytosolic and plastid isoenzymes.