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It is shown that the degree of regulatory kinetic behavior of rabbit muscle phosphofructokinase increases at a given pH and lower temperatures, as well as at a given temperature and lower pH values. It is also shown that the regulatory kinetic behavior which appears at lower pH values is inherent in the tetrameric (active) form of the enzyme. We conclude that a portion of the mechanism proposed previously (Bock, P.E., and Frieden, C. (1976) J. Biol. Chem. 251, 5630-5636) to describe the pH and temperature-dependent inactivation or reactivation may also be used to explain the pH and temperature-dependent regulatory kinetic behavior. According to this proposal, two rapidly equilibrating forms of the enzyme, which differ in the degree of protonation of specific residues, differ in their ability to bind substrates. While the protonated form of the enzyme subsequently becomes inactive by isomerization and dissociation, this process is too slow to affect the kinetic results, making direct comparisons between the association-dissociation behavior and regulatory kinetic behavior invalid. The time dependence of the processes of inactivation or reactivation in the presence or absence of ligands and of the appearance of regulatory kinetic behavior is discussed in relation to their possible role in metabolic regulation.
The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase have been studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. From the enzyme concentration dependence, we conclude that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of one-half 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 four species (two containing four subunits and two containing two 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.