A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).
4-Oxalocrotonate tautomerase (4-OT) is a multimeric, bacterial enzyme comprised of 6 identical 62-amino acid subunits, which associate under native conditions to form a homo-hexameric structure stabilized entirely by noncovalent interactions. We have previously shown that the GuHCl-induced equilibrium unfolding of 4-OT at pH 8.5 is well modeled as a two-state process involving only hexamer and unfolded monomer; and we have obtained spectroscopic evidence that intermediate state(s) is (are) populated in the equilibrium unfolding reaction at pHs 6.0 and 7.4 [Silinski, P., Allingham, M. J., and Fitzgerald, M. C. (2001) Biochemistry 40, 4493-4502]. Here, we report on the pH-induced equilibrium unfolding of 4-OT using size-exclusion chromatography (SEC), far-UV-circular dichroism (CD) spectroscopy, and catalytic activity measurements over the pH range from 1.5 to 10.1. Our results indicate that the native hexamer of 4-OT is the predominant species in solution at pHs > or =6.2, that a partially folded dimeric state of 4-OT is stabilized in solution at pH 4.8, and that the enzyme is largely denatured in strongly acidic solutions (pH < or =3.1). GuHCl-induced equilibrium unfolding studies on 4-OT at pH 4.8 indicate that the folded 4-OT dimer populated at this pH is stabilized by 11.7 kcal.mol(-1). The results of biophysical studies on a fluorescent analogue of the enzyme, 4-OT(F50Y), and the results of UV photo-cross-linking studies on a synthetically derived 4-OT analogue, 4-OT(P1Bpa), suggest the polypeptide chains in the 4-OT dimer are nativelike in structure with the exception of their C-termini.
Silinski
Peter
P
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Fitzgerald
Michael C
MC
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014466:Ultraviolet Rays
C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D019791:Guanidine