Thermodynamic and kinetic characterization of the association of triosephosphate isomerase: the role of diffusion.
It is known that diffusion plays a central role in the folding of small monomeric proteins and in the rigid-body association of proteins, however, the role of diffusion in the association of the folding intermediates of oligomeric proteins has been scarcely explored. In this work, catalytic activity and fluorescence measurements were used to study the effect of viscosity in the unfolding and refolding of the homodimeric enzyme triosephosphate isomerase from Saccharamyces cerevisiae. Two transitions were found by equilibrium and kinetic experiments, suggesting a three-state model with a monomeric intermediate. Glycerol barely affects DeltaG(0)(fold) whereas DeltaG(0)(assoc) becomes more favourable in the presence of the cosolvent. From 0 to 60% (v/v) glycerol, the association rate constant showed a near unitary dependence on solvent viscosity. However, at higher glycerol concentrations deviations from Kramers theory were observed. The dissociation rate constant showed a viscosity effect much higher than one. This may be related to secondary effects such as short-range glycerol-induced repulsion between monomers. Nevertheless, after comparison under isostability conditions, a slope near one was also observed for the dissociation rate. These results strongly suggest that the bimolecular association producing the native dimer is limited by diffusional events of the polypeptide chains through the solvent.
Nájera
Hugo
H
Area Académica de Nutrición, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo, México. hnajera@uaeh.edu.mx
Dagdug
Leonardo
L
Fernández-Velasco
D Alejandro
DA
D004058:Diffusion; D019281:Dimerization; D019277:Entropy; D005990:Glycerol / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*
D029701:Saccharomyces cerevisiae Proteins; D014305:Triose-Phosphate Isomerase; D005990:Glycerol