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 Saccharomyces cerevisiae were investigated by application of kinetic, hydrodynamic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant. Inactivation of the enzyme starts at about 0.3 M GdmCl and undergoes a sharp unfolding transition in a narrow range of the denaturant concentration. 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 circular dichroism and intrinsic fluorescence. Protein aggregates were observed from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations. Refolding of chemically denatured phosphofructokinase proceeds as a stepwise process via the generation of elements of secondary structure, the formation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers. The assembly reactions seem to be rate-limiting. Recovery of the enzyme activity (maximum 65%) competes with an nonproductive aggregation of the subunits. alpha-Cyclodextrin functions as an artificial chaperone by preventing aggregation of the subunits, whereas ATP is suggested to support the generation of heterodimers that are competent to a further assembly.
Bär
J
J
Institut für Biochemie, Universitätsklinikum, Universität Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Golbik
R
R
Hübner
G
G
Kopperschläger
G
G
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003505:Cyclodextrins / Q000494:pharmacology; D019281:Dimerization; D004789:Enzyme Activation; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D009391:Nephelometry and Turbidimetry; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D047391:alpha-Cyclodextrins
D003505:Cyclodextrins; D018832:Molecular Chaperones; D047391:alpha-Cyclodextrins; D010732:Phosphofructokinase-1; D019791:Guanidine; C032613:alpha-cyclodextrin