P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Cooperative interactions between native and modified subunits of rabbit liver fructose 1,6-bisphosphatase

Rabbit liver fructose 1,6-bisphosphatase is converted by subtilisin to a form with smaller subunits and modified catalytic and allosteric properties. Analysis of the changes in the catalytic properties of the enzyme during digestion with subtilisin indicates that these properties depend on the presence of strong functional interactions between all four subunits in the molecule. On the other hand, sensitivity to inhibition by AMP appears to depend only on intrachain interactions. Changes in subunit interaction relating to relaxation in protein conformation during digestion with subtilisin were also inferred from the changes in concentration dependency for the effects of urea on the fluorescent emission spectrum. Structural changes around the region containing the single tryptophan residue appear to be related to the changes in catalytic properties.     

Activation of rabbit kidney fructose diphosphatase by Mg-EDTA, Mn-EDTA and Co-EDTA complexes

Purified rabbit kidney fructose diphosphatase requires both a free cation and a metal-chelate when assayed at pH 8 or below. In the presence Mg²⁺ or Mn²⁺, effective metal chelates were Mn(II)-EDTA, Mg(II)-EDTA, and Co(III)-EDTA. With Mg²⁺ as the cation the affinity of the enzyme for Mn(II)-EDTA or Mg(II)-EDTA was approximately the same, and 300-fold greater than that for Co(III)-EDTA.Activation of the enzyme by the very stable Co(III)-EDTA complex, as well as failure of an ionophore antibiotic to replace EDTA as activator, exclude the possibility that the effects of EDTA are due to removal of metal inhibitors.Inhibition of fructose diphosphatase by Ca²⁺ was competitive with Mg²⁺, and noncompetitive with Mg(II)-EDTA, or Co(III)-EDTA. Conversely inhibition by Zn(II)-EDTA was competitive with Mg(II)-EDTA and noncompetitive with free Mg²⁺. The data suggest that the free metals bind to one site on the enzyme while the metal-EDTA chelates bind to a second site.     

Selective modification of rabbit liver fructose bisphosphatase

Chemical modification of rabbit liver fructose 1,6-bisphosphatase by 5,5′-dithiobis-(2-nitrobenzoic acid) results in thiolation of four highly reactive sulfhydryl groups and a diminished sensitivity to AMP inhibition but not loss of enzyme activity. Ethoxyformylation of the histidine groups of fructose 1,6-bisphosphatase does not result in a sharp loss of activity until at least 4 or 5 of the 13 residues have reacted. Exhaustive formylation does abolish the enzyme's activity. These four most reactive sulfhydryl groups and the one or two least easily modified histidine moieties (those responsible for activity) can be protected against modification by fructose-1,6-P₂ and to a lesser extent by fructose-6-P. The binding of fructose-1,6-P₂ to fructose 1,6-bisphosphatase, however, depends on the presence of structural metal ion since EDTA which removes all endogenous Zn²⁺ from the protein prevents binding of fructose-1, 6-P₂ to the enzyme.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Isotope-tapping experiments with rabbit liver fructose bisphosphatase.

Isotope-trapping experiments with mental-free rabbit liver fructose 1,6-bisphosphatase have shown that enzyme-bound D-fructose 1,6-bisphosphate completely dissociates prior to enzyme turnover initiated by Mn2+ as the catalytic metal. The exchange rate of the binary enzyme-D-fructose 1,6-bisphosphate complex with the substrate pool is, therefore, more rapid than its conversion to products, suggesting that structural Mn2+ is necessary for productive substarate binding. Rapid-quench isotope-trapping experiments confirm the requirement for structural Mn2+ ions for productive binding to occur. These experiments also show that an ordered formation of the enzyme-Mn2+ s-D-fructose 1,6-bisphosphate ternary complex which features metal-ion addition prior to substrate constitutes a catalytically competent pathway in the mechanism of fructose 1,6-bisphosphatase and that all four subunits are active in a single turnover event.     

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.