PHOSPHOFRUCTOKINASE .1. MECHANISM OF PH-DEPENDENT INACTIVATION AND REACTIVATION OF RABBIT MUSCLE ENZYME

  • 1 January 1976
    • journal article
    • research article
    • Vol. 251  (18) , 5630-5636
Abstract
The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase were studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. The enzyme concentration dependence indicates that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of 1/2 the molecular weight. Other data indicate a subsequent isomerization of the dissociated form. The pH and temperature dependence of the inactivation process shows that it is controlled by ionizable groups, and that the apparent pK for these groups is temperature-dependent in such a way as to make the enzyme show the characteristic of cold lability below pH 7. Reactivation of the inactive enzyme occurs by a kinetically different pathway involving deprotonation of an inactive, dissociated form to a form which may either isomerize to another inactive form, or dimerize to the active enzyme. A general mechanism is postulated in which the inactivation and reactivation processes are different aspects of the same mechanism. This mechanism assumes 4 species (2 containing 4 subunits and 2 containing 2 subunits), each of which can exist in a protonated and unprotonated form. Inactivation or reactivation induced by changes in pH or temperature reflect the kinetic establishment of a new steady state between these forms. How the apparent pK values which control the distribution of the enzyme between protonated and unprotonated forms describe the pH-dependent characteristics of the enzyme is discussed in terms of the proposed mechanism.