The Role of the Allosteric Sites in the X‐Ray Inactivation of Phosphorylase b

Crystalline rabbit muscle phosphorylase b was irradiated in dilute aqueous solution with X‐rays. The enzyme was inactivated with a G‐value of 0.09. Measurements of the K m values of the substrate, glucose‐1‐phosphate, and the allosteric activator, adenosine‐5′‐phosphate, demonstrated that these increased linearly with increasing radiation dose. The effect on the K m for the activator was 4 times greater than that on the K m for the substrate. The data indicate that the allosteric function is more sensitive to inactivation than the catalytic function. The enzyme SH‐groups were destroyed by X‐rays with a G‐value of 1.8. Comparison with data on the inactivation of the enzyme by sulfhydryl blocking agents demonstrated that the X‐ray destruction of sulfhydryl groups was sufficiently large to account for the X‐ray inactivation of the enzyme. Blocking of two SH‐groups with p CMB reduced the radiosensitivity of the enzyme by a factor of 2. Measurement of the K m values showed that the p CMB blocking protected preferentially the allosteric sites. The data indicate that the inactivation of phosphorylase b, both by sulfhydryl agents and by X‐rays, involves largely an effect on the allosteric sites with loss of ability to bind the essential activator and consequent loss of ability to bind substrate.