Regulation of pea seed pyrophosphate-dependent phosphofructokinase: Evidence for interconversion of two molecular forms as a glycolytic regulatory mechanism

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Two molecular forms of pyrophosphate-dependent phosphofructokinase (PPi-PFK; pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase; EC 2.7.1.90) have been found whose activity depends upon association and dissociation characteristics regulated by fructose 2,6-bisphosphate (Fru-2,6-P2). PPi-PFK was purified 200-fold from cotyledons of germinating pea seeds and found to exist in two interconvertible molecular forms. The two forms of PPi-PFK have sedimentation coefficients of 6.3 and 12.7 S during ultracentrifugation in sucrose density gradients and also differ both in sensitivity to the activator Fru-2,6-P2 and in affinity for the substrate fructose 6-phosphate. The major component of enzyme activity is in the large form (12.7 S), but the small, less-active, form (6.3 S) predominates when the enzyme preparation is extracted and stored in buffer without Fru-2,6-P2 and glycerol. Urea (1 M) or pyrophosphate (20 mM) treatment results in at least a 50% loss of activity in the glycolytic direction, whereas these treatments had much less influence on the gluconeogenic direction activity. Concomitant with the loss of glycolytic activity the enzyme dissociates into the small form. Fru-2,6-P2 stabilizes the large form of the enzyme against the dissociating effects of pyrophosphate and prevents the inactivation in the glycolytic direction during either urea or pyrophosphate treatment. The small molecular form of the enzyme is converted into the large form in the presence of Fru-2,6-P2. We propose that glycolytic and gluconeogenic hexose metabolism in plants includes a regulatory mechanism induced by Fru-2,6-P2 that involves the interconversion of two molecular forms of PPi-PFK.

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