Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants.
Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.
Ludwig
Heide C
HC
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Pardo
Fabian N
FN
Asenjo
Joel L
JL
Maureira
Marco A
MA
Yañez
Alejandro J
AJ
Slebe
Juan C
JC
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine; D014364:Tryptophan / Q000235:genetics*
D000817:Anilino Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D014364:Tryptophan; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D019791:Guanidine