SUBSTRATE-INDUCED REACTIVATION OF SPINACH RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE DENATURED BY LOW CONCENTRATIONS OF GUANIDINE-HYDROCHLORIDE/

The unfolding and refolding behavior of the hexadecameric ribulose-1,5 -bisphosphate carboxylase/oxygenase (Rubisco) from spinach in solution s of guanidine hydrochloride (GdnHCl) was studied. By a number of crit eria (enzyme activity, protein fluorescence, circular dichroism), the enzyme was judged to be almost completely unfolded in 6M GdnHCl. The c hanges 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 intrinsi c fluorescence. The result of the size-exclusion chromatography indica tes 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-dep endent and obeyed first-order kinetics, and the substrate, ribulose-1, 5-biphosphate, can stimulate this reactivation process. The result sug gests that the inactivation of the Rubisco in dilute GdnHCl is caused by the conformational changes at the active site instead of the inhibi tion of guanidine hydrochloride or the dissociation of the oligomeric enzyme molecules. (C) 1997 Elsevier Science B.V.