Reversible unfolding of fructose 6-phosphate, 2-kinase

fructose 2,6- bisphosphatase

N. Tominaga, D. M. Jameson, K. Uyeda

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino)ethyl)naphthalene-1- sulfonic acid) probes. The unfolding reaction is described minimally as a 4- state transition from folded dimer → partially unfolded dimer → monomer → unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.

Original languageEnglish (US)
Pages (from-to)1245-1252
Number of pages8
JournalProtein Science
Volume3
Issue number8
StatePublished - 1994

Fingerprint

Phosphofructokinase-2
Tryptophan
Dimers
Acrylamide
Quenching
Iodides
Phosphotransferases
Monomers
Enzyme activity
Phosphoric Monoester Hydrolases
Enzymes
Fluorescence
Peptides
Sulfonic Acids
Guanidine
Shielding
Testis
Rats
Membrane Proteins
Adenosine Triphosphate

Keywords

  • fluorescence quenching studies
  • fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase
  • liver
  • muscle
  • reversible unfolding

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reversible unfolding of fructose 6-phosphate, 2-kinase : fructose 2,6- bisphosphatase. / Tominaga, N.; Jameson, D. M.; Uyeda, K.

In: Protein Science, Vol. 3, No. 8, 1994, p. 1245-1252.

Research output: Contribution to journalArticle

Tominaga, N. ; Jameson, D. M. ; Uyeda, K. / Reversible unfolding of fructose 6-phosphate, 2-kinase : fructose 2,6- bisphosphatase. In: Protein Science. 1994 ; Vol. 3, No. 8. pp. 1245-1252.
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