Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants

Ludwig, HC; Pardo, FN; Asenjo, JL; Maureira, MA; Yanez, AJ; Slebe, JC

Abstract

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 ∼ 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. © 2007 The Authors.

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Título según WOS: Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants
Título según SCOPUS: Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants
Título de la Revista: FEBS JOURNAL
Volumen: 274
Número: 20
Editorial: WILEY-BLACKWELL
Fecha de publicación: 2007
Página de inicio: 5337
Página final: 5349
Idioma: English
URL: http://doi.wiley.com/10.1111/j.1742-4658.2007.06059.x
DOI:

10.1111/j.1742-4658.2007.06059.x

Notas: ISI, SCOPUS