Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state.
Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.
D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D006146:Guanidines; D046911:Macromolecular Substances; D005969:Glutamate Dehydrogenase