TY - JOUR
T1 - Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae
T2 - Different mechanisms, similar activation enthalpies
AU - Moreno-Vargas, Liliana M.
AU - Carrillo-Ibarra, Normandé
AU - Arzeta-Pino, Lilian
AU - Benítez-Cardoza, Claudia G.
N1 - Funding Information:
This work was supported by grants from TWAS , CONACyT ( 45990 and 101229 ), ICyT-DF ( 6968 ), and SIP-IPN ( 20111186 ).
PY - 2011/12/1
Y1 - 2011/12/1
N2 - Yeast enolase is stabilized by its natural cofactor Mg2+. This stabilization is ascribed to the reduced subunit dissociation of the holoprotein. Nevertheless, how Mg2+ alters the unfolding mechanism has yet to be fully characterized. Here, we investigate the role of Mg2+ in the denaturation mechanism and unfolding kinetics of yeast enolase. Apo-enolase unfolds through a three-state process (N2↔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 (N2→2D). Our results indicate smaller unfolding rate-constants in the presence of Mg2+, 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 (185-190kJmol-1).
AB - Yeast enolase is stabilized by its natural cofactor Mg2+. This stabilization is ascribed to the reduced subunit dissociation of the holoprotein. Nevertheless, how Mg2+ alters the unfolding mechanism has yet to be fully characterized. Here, we investigate the role of Mg2+ in the denaturation mechanism and unfolding kinetics of yeast enolase. Apo-enolase unfolds through a three-state process (N2↔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 (N2→2D). Our results indicate smaller unfolding rate-constants in the presence of Mg2+, 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 (185-190kJmol-1).
KW - Circular dichroism
KW - Cofactor
KW - Dimer
KW - Intermediate state
KW - Irreversibility
KW - Molten globule
KW - Monomer
KW - Thermal unfolding kinetics
UR - http://www.scopus.com/inward/record.url?scp=80054847243&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2011.07.021
DO - 10.1016/j.ijbiomac.2011.07.021
M3 - Artículo
C2 - 21893090
AN - SCOPUS:80054847243
SN - 0141-8130
VL - 49
SP - 871
EP - 878
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
IS - 5
ER -