Stimulation of Fructose 1,6-Diphosphatase by Sulfhydryl Reagents and Interaction between the Different Enzymic Sites in the Molecule

The mechanism of the activation of rabbit liver fructose 1,6-diphosphatase by sulfhydryl reagents has been studied. Per sulfhydryl group modified, different sulfhydryl reagents stimulated the enzyme to different extents. Disulfides increased the enzyme activity by 80–90% per sulfhydryl group blocked, while the value for the other reagents tested was 15–25%. With all reagents used the stimulation increased linearly with SH blocking until 5–6 sulfhydryl groups had been modified. Further blocking of SH groups led to progressive loss of enzyme activity. Enzymes which had been stimulated by blocking of 5–6 SH groups by 5,5′-dithio-bis(2-nitrobenzoic acid) or para-chloromercuribenzoate had properties different from the native enzyme. The inhibition by AMP and by substrate was increased. Unlike the native enzyme, the disulfide modified enzyme precipitated at pH below 4. The remaining free sulfhydryl groups in the two modified enzymes showed different reactivities, and blocking of these SH groups with para-chloromercuribenzoate reduced the activity to different extents in the two cases. The data indicate that the stimulation of fructose-1,6-diphosphatase by blocking of sulfhydryl groups is due to conformational changes of the protein. Inhibition of the enzyme by AMP was found to increase with increasing substrate concentration. It is suggested that this may be of significance in the regulatory function of the enzyme.