X-ray modification of the catalytic and allosteric functions of fructose-1,6-diphosphatase

Abstract

1. 1. Native rabbit liver fructose-1,6-diphosphatase ( d-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11) and enzyme stimulated by disulfide formation have been irradiated in solution with X-rays. 2. 2. The dose-response curve for the native enzyme showed an initial stimulation of catalytic activity, followed by subsequent exponential inactivation. The disulfide-modified enzyme was inactivated exponentially with no initial stimulation. The G values for inactivation of catalytic activity were 0.017 and 0.009 for the native and the disulfide-modified enzymes, respectively. 3. 3. The allosteric activity of the native enzyme, as measured by the inhibition by AMP, was much more radioresistant than the catalytic activity. In the modified enzyme, these two functions were of comparable radiosensitivity. 4. 4. The ability of the enzymes to be stimulated by mixed disulfide formation was about 10 times as sensitive to X-ray inactivation as was the catalytic function. The loss of sensitivity to stimulation by disulfides was due to destruction of 5–6 specific SH groups which were particularly radiosensitive ( G value, 1.2). 5. 5. The X-ray-induced reduction in enzyme activity was associated with destruction of enzyme SH groups of low radiosensitivity ( G value, 0.07. The loss of enzyme activity was partly reversed by addition of cysteamine after exposure. The results indicate that sulfhydryl groups play an important, but not exclusive role in the X-ray inactivation of fructose-1,6-diphosphatase. 6. 6. The presence of AMP during irradiation protected the enzyme almost completely against inactivation of the allosteric function. The results indicated that the X-ray inactivation of the allosteric function of fructose-1,6-diphosphatase is due to a specific modification of the binding sites for AMP.