Limited Proteolysis of Yeast Phosphofructokinase by Subtilisin

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Abstract
Yeast phosphofructokinase having a molecular weight of 750000–800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits α and β (Mr 120000) are converted to α′ and β′ (Mr approximately 90000), and in the second step, accompanied by a decrease in enzyme activity, α′ is converted to α″ (Mr 80000) and two further fragments having Mr 45000 and 35000 become detectable. In the course of this conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of α′ and β′, whereas ATP prevents only degradation of β′. When in presence of ATP α′ is degraded to α″, the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of this molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of α″ to β′ is one-to-one.