title: The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant.
abstract: 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 (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.
authors.forename: Edgar
authors.initials: E
authors.lastname: Deu
authors.forename: Jack F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D001051:Apoenzymes / Q000737:chemistry*; D001219:Aspartate Aminotransferases / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D019791:Guanidine / Q000494:pharmacology*; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000494:pharmacology*
chemical_list: D001051:Apoenzymes; D014508:Urea; D001219:Aspartate Aminotransferases; D019791:Guanidine