A Stable Dimer in the pH-Induced Equilibrium Unfolding of the Homo-Hexameric Enzyme 4-Oxalocrotonate Tautomerase (4-OT)

• Published in: Biochemistry (Easton) 2002-04, Vol.41 (13), p.4480-4491
• Main Authors: Silinski, Peter, Fitzgerald, Michael C
• Format: Article
• Language: English
• Subjects: Amino acids; Catalysis; Chromatography, High Pressure Liquid; Circular Dichroism; Crosslinking; Dimerization; Dimers; Dose-Response Relationship, Drug; Guanidine - pharmacology; Hydrogen-Ion Concentration; Isomerases - chemistry; Kinetics; Mass Spectrometry; Models, Biological; Models, Chemical; Monomers; pH effects; Polypeptides; Protein Denaturation; Protein Folding; Size exclusion chromatography; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Ultraviolet Rays
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi011872w

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 ≥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 ≤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.