Pihl A: Modification of regulatory properties of phosphofructokinase acetylation.

Abstract

The effect of acetylation and of sulfhydryl blocking agents on phosphofructokinase from rabbit muscle has been studied. Treatment of phosphofructokinase with N-acetylimidazole led to rapid loss of its allosteric function, as measured by the ability of AMP to stimulate the ATP-inhibited enzyme. The catalytic activity was far less sensitive to acetylation. The loss of allosteric function on acetylation was due to the fact that the modified enzyme was insensitive to ATP inhibition. The presence of ATP during acetylation prevented the loss of allosteric activity. The results indicate that tyrosine residues may play an important role in the inhibitory binding sites of ATP. Incubation of phosphofructokinase with a 50-fold excess of para-chloromercuribenzoate led to complete inactivation of the enzyme, an effect which could be reversed by the addition of excess thiol. Sulfhydryl blocking had no effect on the ability of AMP to stimulate the ATP-inhibited enzyme. The results confirm that sulfhydryl groups are closely associated with the catalytic site of phosphofructokinase, and demonstrate that they are not involved in the inhibition of the enzyme by ATP and its reversal by AMP.