Reversible unfolding of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase

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-(iodoacety1)amino) ethy1)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.

Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase

“…In contrast to the current results, previous data (Tominaga et al, 1994) showed that Fru 6-P,Z-kinase was inactivated below 0.5 M guanidine. This difference was attributable to the enzymes being stored frozen for more than a few weeks.…”

“…3), significant unfolding of Trp 299, Trp 320, and Trp 15 occurred in dilute guanidine (10.5 M), even though both enzyme remained active, but no unfolding of Trp 64 and the wild-type enzymes was observed under the same conditions. Sharp transition during unfolding was observed in the latter enzymes only when guanidine was increased from 0.5 to 1 M. The previous fluorescent polarization measurements (Tominaga et al, 1994) demonstrated that the enzyme dissociates in the same range of guanidine, which is closely associated with inactivation of both enzymes (Fig. 3A,C).…”

“…However, the intensity of the latter enzymes dropped sharply between 0.5 M and 0.8 M guanidine, and more gradually above these guanidine concentrations. These results may indicate that the regions of Trp 64 and Trp 15 in the kinase domain began to unfold as soon as the enzyme was exposed to dilute guanidine, but those regions in the phosphatase domain were stable until guanidine concentration exceeded 0.5 M. The previous results with the DANS-labeled wild-type enzyme indicated that the fluorescence polarization changes drastically between 0.7 M and 2 M quanidine in protein concentration-dependent manner, indicating dissociation of dimer to monomer (Tominaga et al, 1994).…”

“…This idea is further supported by other circumstantial evidence, Deletion of the N-terminal peptide of the enzyme increases K,,, for Fru 6-P, and the deleted enzyme is more susceptible to dissociation and inactivation (Tominaga et al, 1993). Fru 6-P protects the enzyme against heat and denaturant inactivation and also enhances the rate of renaturation of guanidine or urea-denatured enzymes (Tominaga et al, 1994). The fact that the polarization of Trp 64 was not affected by Fru 6-P suggests that this Trp residue is not located near the Fru 6-P binding site.…”

“…These results were further analyzed using the mod- We should note that Fu plot presumes a two-state unfolding model. We realize the unfolding-refolding of the enzyme involves more than two states of the enzyme conformation (Tominaga et al, 1994). However, Fu equation was used for comparison in normalizing the denaturation curves of these mutants.…”