Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.
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.
Tominaga
N
N
Research Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
Jameson
D M
DM
Uyeda
K
K
D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D005454:Fluorescence Polarization; D005636:Fructosephosphates / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007454:Iodides / Q000494:pharmacology; D046911:Macromolecular Substances; D008297:Male; D009097:Multienzyme Complexes / Q000737:chemistry*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D010770:Phosphotransferases / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013737:Testis / Q000201:enzymology
D000178:Acrylamides; D005636:Fructosephosphates; D006146:Guanidines; D007454:Iodides; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D019791:Guanidine