The unfolding pathway for apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant

The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (m(D*reversible arrow U) = 3.4 +/- 0.3 kcal mol(-1) M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D reversible arrow 2M reversible arrow 2M* reversible arrow 2U in GdnHCl and D reversible arrow D* reversible arrow 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D reversible arrow 2U) is 36 +/- 3 kcal mol(-1), while that for the D* reversible arrow 2U transition is 24 +/- 2 kcal mol(-1), both at 1 M standard state and pH 7.5.