On the Mechanism of Action of Fructose 1,6-Diphosphatase. Inhibition by Structural Analogs of Fructose 1,6-Diphosphate

Based on the behavior of the substrate analogs, α-methyl-D-fructofuranoside 1,6-diphosphate (II), 2,5-anhydro-D-glucitol 1,6-diphosphate (III), β-methyl-D-fructofuranoside 1,6-diphosphate (IV), and 2,5-anhydro-D-mannitol 1,6-diphosphate (V), toward fructose 1,6-diphosphatase (FDPase) at pH 9.4 the following conclusions have been reached: (1) that the furanose form of the sugar diphosphate is the active configuration since binding constants determined kinetically and by direct measurement from fluorescence studies are identical with those of fructose 1,6-diphosphate (FDP); (2) that the C-3 and C-4 hydroxyls of the furanose ring are essential since cis-2,-5-bis(hydroxymethyl)tetrahydrofuran diphosphate did not kinetically assay as a substrate or an inhibitor; (3) that all the analogs, II- V, inclusive, at concentration <10^-3 M act as competitive inhibitors occupying the active site of FDPase; (4) that a change in kinetic behavior as a function of β configuration (IV, V) at high concentrations (>10^-3 M) is observed with the enzyme and may be related to inhibition of the enzyme by its own substrate. In general, inhibition of allosteric enzymes by their own substrates may be found in cases where the substrate occuss in two rapidly equilibrating configurations, i.e., the α and the β anomers of FDP. Data from acetylation experiments at pH 7.5 suggest the possibility of interactions between the tyrosyl residues of FDPase and the 2-OH position of FDP.