Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae: different mechanisms, similar activation enthalpies.

Yeast enolase is stabilized by its natural cofactor Mg(2+). This stabilization is ascribed to the reduced subunit dissociation of the holoprotein. Nevertheless, how Mg(2+) alters the unfolding mechanism has yet to be fully characterized. Here, we investigate the role of Mg(2+) in the denaturation mechanism and unfolding kinetics of yeast enolase. Apo-enolase unfolds through a three-state process (N(2)↔2I→2D). The intermediate species is described as a monomeric molten globule-like conformation that becomes noticeable in the presence of phosphate and is able to recover its native secondary structure when cooled down. Kinetic studies confirmed the presence of the intermediate species, even though it was not noticeable in the thermal scans. The cofactor increases the cooperativity of the unfolding transitions, while the intermediate species becomes less noticeable or nonexistent. Thus, holo-enolase follows a simple two-state mechanism (N(2)→2D). Our results indicate smaller unfolding rate-constants in the presence of Mg(2+), thus favoring the native state. The temperature dependence of the unfolding rates allowed us to calculate the activation enthalpies of denaturation. Interestingly, despite the different unfolding mechanisms of the apo and holo forms of enolase, they both have similar activation barriers of denaturation (185-190 kJ mol(-1)).

Chemical unfolding of enolase from Saccharomyces cerevisiae exhibits a three-state model.

Enolase is a multifunctional protein that participates in glycolysis and gluconeogenesis and can act as a plasminogen receptor on the cell surface of several organisms, among other functions. Despite its participation in a variety of biological and pathophysiological processes, its stability and folding/unfolding reaction have not been fully explored. In this paper we present, the urea and GdnHCl-induced denaturation of enolase studied by means of fluorescence and circular dichroism spectroscopies. We found that enolase unfolds through a highly reversible pathway, populating a stable intermediate species in a range of experimental conditions. The refolding reaction also exhibits an intermediate state that might have a slightly more compact conformation compared to the unfolding intermediate. The thermodynamic parameters associated with the unfolding reaction are presented and discussed.