title: Substrate induced reactivation of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase denatured by low concentrations of guanidine hydrochloride.
abstract: The unfolding and refolding behavior of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach in solutions of guanidine hydrochloride (GdnHCl) was studied. By a number of criteria (enzyme activity, protein fluorescence, circular dichroism), the enzyme was judged to be almost completely unfolded in 6 M GdnHCl. The changes in enzyme activity occur at lower concentrations of GdnHCl than those required to bring about changes in circular dichroism (CD) and fluorescence, as has been found for other enzymes. Spinach Rubisco is completely inactive in 0.5 M GdnHCl with no apparent changes observed in the overall structure of the enzyme as monitored by CD and intrinsic fluorescence. The result of the size-exclusion chromatography indicates that the inactive enzyme still exists in the hexadecameric state. On dilution of the GdnHCl, reactivation of the inactive enzyme by low concentrations of GdnHCl occurred. The regain of activity was time-dependent and obeyed first-order kinetics, and the substrate, ribulose-1,5-biphosphate, can stimulate this reactivation process. The result suggests that the inactivation of the Rubisco in dilute GdnHCl is caused by the conformational changes at the active site instead of the inhibition of guanidine hydrochloride or the dissociation of the oligomeric enzyme molecules.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
authors.forename: R F
authors.initials: RF
authors.lastname: Jiang
authors.forename: Z X
authors.initials: ZX
authors.lastname: Wang
authors.forename: G J
authors.initials: GJ
authors.lastname: Xu
mesh_terms: D004789:Enzyme Activation; D019791:Guanidine / Q000378:metabolism*; D007700:Kinetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D018724:Spinacia oleracea / Q000201:enzymology*
chemical_list: D012273:Ribulose-Bisphosphate Carboxylase; D019791:Guanidine