Lanthanide pyrophosphates as substrates for the pyrophosphate-dependent phosphofructokinases from Propionibacterium freudenreichii and Phaseolus aureus: evidence for a second metal ion required for reaction.
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 microM), both enzymes will utilize a variety of metal-pyrophosphate complexes as reactant in the direction of fructose 6-phosphate (F6P) phosphorylation. The Vmax values are about 100-fold lower and the Km 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 Km values of the above remain essentially unchanged, while Vmax 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 microM 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 microM. The exchange-inert CrIII(PPi)(H2O)4 complex is not a substrate, but is an inhibitor competitive against MgPPi with a Ki of 27 microM. Results are discussed in terms of the possible role of the divalent metal ion activators.[1]References
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