Binding and kinetic data for rabbit liver fructose-1,6-bisphosphatase with Zn2+ as cofactor.
Atomic absorption determinations of zinc content were employed to demonstrate the technique to obtain zinc-free rabbit liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). Reactivation of the apoenzyme by Zn(2+) is rapid (within 1 min) and restores up to 96% of the initial specific activity. Gel filtration measurements showed that the enzyme contains four binding sites for zinc per molecule, one per subunit. The dissociation constants for the initial two binding sites are less than 0.1 muM. In the presence of a substrate analog, (alpha + beta) methyl D-fructofuranoside 1,6-bisphosphate, at a level where two analog molecules are bound per phosphatase molecule, a total of eight Zn(2+) ions bind at 8 muM Zn(2+), revealing the presence of additional binding sites, including the catalytic one. The activity in the presence of Zn(2+) is maximal at ca. 8 muM Zn(2+), which corresponds to saturation of the four subunit sites plus the catalytic sites in the presence of substrate. At metal ion concentrations less than 10 muM, the order of activation is Zn(2+) > Mn(2+) > Mg(2+). In kinetic assays with two metal cofactors the effect of Zn(2+) at concentrations less than 10 muM on either the Mg(2+) or the Mn(2+) assays is inhibitory owing to the apparent formation of mixed (two different elements) metal ion-enzyme complexes possessing a catalytic activity that is measureable but lower than anticipated if the catalysis by the various metal ions is simply additive. Hence the activation by EDTA of the Mg(2+) and Mn(2+) assays is explicable in terms of Zn(2+) removal, thus eliminating mixed metal species. Collectively these observations suggest that fructose-1,6-bisphosphatase may function in vivo as a Zn(2+) metalloprotein.
D000818:Animals; D001051:Apoenzymes; D001665:Binding Sites; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D015032:Zinc / Q000378:metabolism*
D001051:Apoenzymes; D002413:Cations, Divalent; D008345:Manganese; D004492:Edetic Acid; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D015032:Zinc