In the absence of Mg2+, both the dimeric bacterial and tetrameric plant fructose 2,6-bisphosphate-activated pyrophosphate-dependent phosphofructokinases (PPi-PFKs) are inactive at pH 8 and 25 degrees C. In the presence of a low concentration of Mg2+ (5 mu M), both enzymes will utilize a variety of metal-pyrophosphate complexes as reactant in the direction of fructose 6-phosphate (F6P) phosphorylation. The V-max values are about 100-fold lower and the K-m values about 10-fold greater than those measured with MgPPi when lanthanide-PPi complexes are used as a substrate. In the presence of added Mg2+, the K-m values of the above remain essentially unchanged, while V-max values increase 10-fold for lanthanide-PPi complexes. These data, along with the 12-16 order of magnitude increased affinity of the lanthanides for PPi compared to Mg2+, indicate that the PPi-PFKs require two metal ions for catalysis, one to form a chelate with PPi and a second as an essential activator. With CePPi, an activation constant of about 25 mu M is measured for Mg2+. In addition, a number of other divalent (but no tripositive) metal ions serve as activators including Mn2+, Co2+, Mo2+, Cr2+, Fe2+, and Ni2+; activation constants are in the range 20-150 mu M. The exchange-inert Cr-III(PPi)(H2O)(4) complex is not a substrate, but is an inhibitor competitive against MgPPi with a K-i of 27 mu M. Results are discussed in terms of the possible role of the divalent metal ion activators.
