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 und...

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Bibliographic Details
Published in:Biochemistry (Easton) 2000-06, Vol.39 (23), p.6960-6968
Main Authors: Bär, Jörg, Golbik, Ralph, Hübner, Gerhard, Kopperschläger, Gerhard
Format: Article
Language:English
Subjects:
1-phosphofructokinase
alpha-Cyclodextrins
Chromatography, Gel
Circular Dichroism
Cyclodextrins - pharmacology
Dimerization
Enzyme Activation
Guanidine - pharmacology
guanidinium chloride
Kinetics
Molecular Chaperones - metabolism
Nephelometry and Turbidimetry
Phosphofructokinase-1 - chemistry
Protein Conformation
Protein Denaturation
Protein Folding
Protein Structure, Secondary
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Spectrometry, Fluorescence
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Summary: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. α-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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi9928142