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.
Bertagnolli
B L
BL
Department of Microbiology and Immunology, University of North Texas Health Science Center at Fort Worth 76107.
Cook
P F
PF
D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008674:Metals, Rare Earth / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010946:Plants, Medicinal; D011424:Propionibacterium / Q000201:enzymology*; D013379:Substrate Specificity
D002413:Cations, Divalent; D005636:Fructosephosphates; D008670:Metals; D008674:Metals, Rare Earth; D010710:Phosphates; D003035:Cobalt; D008345:Manganese; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1; D008274:Magnesium