Phosphofructokinase from baker's yeast: properties of a proteolytically modified active enzyme form

Abstract

The structural and functional stability of an active fragment of yeast phosphofructokinase (called 12 S-enzyme) obtained by limited proteolysis was investigated. In the course of proteolysis the subunits of the native enzyme (α and β) are partially degraded to forms of lower molecular weight (designated as α' and β') which is accompanied by a dissociation of the native octamer to tetramers. Upon dilution the tetramic 12 S-enzyme becomes inactivated as a consequence of its dissociation. Hydrodynamic studies suggest a two-step dissociation mechanism with the formation of dimers and monomers. Inactivation of the enzyme fails or is retarded at millimolar concentrations of either fructose 6-phrosphate or ATP. Sulphate and phosphate have a similar effect but at molar concentrations. The latter two anions are also able to reactivate the inactivated enzyme. This reactivation is not due to an increase of the ionic strength but seems to be rather specifically caused by the lyotropic properties of sulphate and phosphate. The reactivation is accompanied by a partial reassociation of the enzyme. An association-dissociation model in the presence of sulphate providing only dimers as smallest species was calculated from the hydrodynamic date. The conclusion can be drawn that the tetramic form of the enzyme is the smallest active fragment of the 12 S-phosphofructokinase.