REVERSIBLE UNFOLDING OF FRUCTOSE 6-PHOSPHATE, 2-KINASE-FRUCTOSE 2,6-BISPHOSPHATASE

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fruct ose 2,6-bisphosphatase in guanidine hydrochloride was monitored by fol lowing enzyme activities as well as by fluorescence methodologies (int ensify, emission maximum, polarization, and quenching), using both int rinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)napht halene-1-sulfonic acid) probes. The unfolding reaction is described mi nimally as a 4-state transition from folded dimer --> partially unfold ed dimer --> monomer --> unfolded monomer. The partially unfolded dime r 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 acti ve enzyme. The recovery rates of the kinase and the phosphatase were s imilar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermedia tes. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, wer e only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosp hate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-t erminal peptide (containing a tryptophan) was not exposed on the prote in surface and may play an important role in shielding other tryptopha ns from solvent.