The existence of a hexameric intermediate with molten-globule-like properties in the thermal denaturation of bovine-liver glutamate dehydrogenase.
We have studied the thermal denaturation of hexameric beef-liver glutamate dehydrogenase by itself and in the presence of ADP and guanidine-HCl by a variety of techniques. In differential scanning calorimetry studies, the observed melting temperature and total enthalpy of denaturation show no dependence on protein concentration, but do show significant dependence on the scan rate. This suggests that the overall denaturation process is irreversible and kinetically controlled. Isothermal unfolding kinetics from spectrophotometry confirm this result. The size of the protein, as shown by quasi-elastic light scattering measurements, does not change during the denaturation process. We interpret these results in terms of the following model: N6 reversible N'6-->6U(-->F) where N6 and N'6 are, respectively, the native hexamer and a hexameric, highly folded high-enthalpy species, U is the unfolded monomer and F is some final aggregated state. The kinetic intermediate, N'6, possesses the properties of one definition of a molten globule, having a very high enthalpy and a hexameric compact structured form. This "molten globule" is an obligatory intermediate in the unfolding pathway of the protein. The stabilization of the protein by ADP is due to the modulation of the high-enthalpy two-state predenaturational E reversible E' transition, resulting in the lowering of the energy of the native state of the protein.
Singh
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Laboratory of Molecular Biochemistry, Veterans Affairs Medical Center, Kansas, MO, USA.
Liu
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Fisher
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase; D019791:Guanidine