Denaturation of phosphofructokinase-1 from Saccharomyces cerevisiae by guanidinium chloride and reconstitution of the unfolded subunits to their catalytically active form

Unfolding and refolding of heterooctameric phosphofructokinase-1 from Sacch aromyces cerevisiae were investigated by application of kinetic, hydrodynam ic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant. Inactivation of the enzyme starts at about 0.3 M GdmCl and und ergoes a sharp unfolding transition in a narrow range of the denaturant con centration. The inactivation is accompanied by a dissociation of the enzyme into dimers (at 0.6 M GdmCl), which could be detected by changes of the ci rcular dichroism and intrinsic fluorescence. Protein. aggregates were obser ved from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations . Refolding of chemically denatured phosphofructokinase proceeds as a stepw ise process via the generation of elements of secondary structure, the form ation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers. The assembly rea ctions seem to be rate-limiting. Recovery of the enzyme activity (maximum 6 5%) competes with an nonproductive aggregation of the subunits. alpha-Cyclo dextrin functions as an artificial chaperone by preventing aggregation of t he subunits, whereas ATP is suggested to support the generation of heterodi mers that are competent to a further assembly.