期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1978
卷号：75
期号：5
页码：2185-2189
DOI：10.1073/pnas.75.5.2185
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 Zn2+ 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 {micro}M. In the presence of a substrate analog, ( + {beta}) methyl D-fructofuranoside 1,6-bisphosphate, at a level where two analog molecules are bound per phosphatase molecule, a total of eight Zn2+ ions bind at 8 {micro}M Zn2+, revealing the presence of additional binding sites, including the catalytic one. The activity in the presence of Zn2+ is maximal at ca. 8 {micro}M Zn2+, 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 {micro}M, the order of activation is Zn2+ > Mn2+ > Mg2+. In kinetic assays with two metal cofactors the effect of Zn2+ at concentrations less than 10 {micro}M on either the Mg2+ or the Mn2+ 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 Mg2+ and Mn2+ assays is explicable in terms of Zn2+ removal, thus eliminating mixed metal species. Collectively these observations suggest that fructose-1,6-bisphosphatase may function in vivo as a Zn2+ metalloprotein.
