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A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).

Publication ,  Journal Article
Silinski, P; Fitzgerald, MC
Published in: Biochemistry
April 2002

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.

Duke Scholars

Published In

Biochemistry

DOI

EISSN

1520-4995

ISSN

0006-2960

Publication Date

April 2002

Volume

41

Issue

13

Start / End Page

4480 / 4491

Related Subject Headings

  • Ultraviolet Rays
  • Time Factors
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Fluorescence
  • Protein Folding
  • Protein Denaturation
  • Models, Chemical
  • Models, Biological
  • Mass Spectrometry
 

Citation

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Silinski, P., & Fitzgerald, M. C. (2002). A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT). Biochemistry, 41(13), 4480–4491. https://doi.org/10.1021/bi011872w
Silinski, Peter, and Michael C. Fitzgerald. “A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).Biochemistry 41, no. 13 (April 2002): 4480–91. https://doi.org/10.1021/bi011872w.
Silinski, Peter, and Michael C. Fitzgerald. “A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).Biochemistry, vol. 41, no. 13, Apr. 2002, pp. 4480–91. Epmc, doi:10.1021/bi011872w.
Journal cover image

Published In

Biochemistry

DOI

EISSN

1520-4995

ISSN

0006-2960

Publication Date

April 2002

Volume

41

Issue

13

Start / End Page

4480 / 4491

Related Subject Headings

  • Ultraviolet Rays
  • Time Factors
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Fluorescence
  • Protein Folding
  • Protein Denaturation
  • Models, Chemical
  • Models, Biological
  • Mass Spectrometry