The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear. It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons. To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast (Saccharomyces cerevisiae) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques. The unfolding and refolding of this enzyme are not coincident after ‘conventional’ equilibrium times. Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process. By contrast, equilibrium was reached in the refolding direction. The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. The Gibbs energy changes for monomer folding (ΔGfold0 = −16.6±0.7kJ·mol-1) and monomer association (ΔGassoc0 = −70.3±1.1kJ·mol-1) were calculated from data obtained in the two denaturants. From an analysis of the present data and data from the literature on the stability of TIM from different species and for other β/α barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM. There is interplay between function and stability.
- dimer evolution
- monomeric intermediate
↵1 On sabbatical leave from the Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria. México, D.F. 04510, México.
Abbreviations used: DTT, dithiothreitol; GdmCl, guanidinium chloride; GDPH, glycerol-3-phosphate dehydrogenase; N→U, native dimer→unfolded monomer; Rs, Stokes radius; SCM, spectral centre of mass; TIM, triosephosphate isomerase; hTIM, human TIM; rTIM, rabbit TIM; TbTIM, Trypanosoma brucei TIM; yTIM, yeast (Saccharomyces cerevisiae) TIM.
- The Biochemical Society, London ©2003