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.