tailieunhanh - Báo cáo khoa học: Unraveling multistate unfolding of pig kidney fructose-1,6bisphosphatase 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 hydrochlo-ride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yan˜ez, A. J., Rodriguez, P. H and Slebe, J. C. | ỊFEBS Journal Unraveling multistate unfolding of pig kidney fructose-1 6-bisphosphatase using single tryptophan mutants Heide C. Ludwig Fabian N. Pardo Joel L. Asenjo Marco A. Maureira Alejandro J. Yanez and Juan C. Slebe Institute de Bioquimica Facultad de Ciencias Universidad Australde Chile Valdivia Chile Keywords fructose-1 6-bisphosphatase protein unfolding single tryptophan mutants tetrameric intermediate phase diagram Correspondence J. C. Slebe Instituto de Bioquimica Universidad Australde Chile Campus Isla Teja Valdivia Chile Fax 56 63 221406 Tel 56 63 221797 E-mail jslebe@ These authors contributed equally to this work Received 18 June 2007 revised 14 August 2007 accepted 21 August 2007 doi 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. Yanez 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 .