Thermodynamic characterization of yeast triosephosphate isomerase refolding : insights into the interplay between function ans stability as reason for the oligomeric nature of the enzyme

Abstract

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 cere.isiae) 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 simples equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. 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.