Binding and kinetic data for rabbit liver fructose-1,6-bisphosphatase with Zn 2+ as cofactor

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Abstract
Atomic absorption determinations of Zn 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 Zn2+ is rapid (within 1 min) and restores up to 96% of the initial specific activity. Gel filtration measurements showed that the enzyme contains 4 binding sites for Zn/molecule, 1/subunit. The Kd for the initial 2 binding sites are less than 0.1 .mu.M. In the presence of a substrate analog, (.alpha. + .beta.) methyl D-fructofuranoside 1,6-bisphosphate, at a level where 2 analog molecules are bound/phosphatase molecule, a total of 8 Zn2+ ions bind at 8 .mu.M Zn2+, revealing the presence of additional binding sites, including the catalytic one. The activity in the presence of Zn2+ is maximal at approximately 8 .mu.M Zn2+, which corresponds to saturation of 4 subunit sites plus the catalytic sites in the presence of substrate. At metal ion concentrations less than 10 .mu.M, the order of activation is Zn2+ > Mn2+ > Mg2+. In kinetic assays with 2 metal cofactors the effect of Zn2+ at concentrations less than 10 .mu.M on either the Mg2+ or the Mn2+ assays is inhibitory owing to the apparent formation of mixed 2 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. The activation by EDTA of the Mg2+ and Mn2+ assays is explicable in terms of Zn2+ removal, thus eliminating mixed metal species. Fructose-1,6-bisphosphatase may function in vivo as a Zn2+ metalloprotein.