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 gluta mate dehydrogenase by itself and in the presence of ADP and guanidine- HCl by a variety of techniques. In differential scanning calorimetry s tudies, the observed melting temperature and total enthalpy of denatur ation show no dependence on protein concentration, but do show a signi ficant dependence on the scan rate. This suggests that the overall den aturation process is irreversible and kinetically controlled. Isotherm al unfolding kinetics from spectrophotometry confirm this result. The size of the protein, as shown by quasi-elastic light scattering measur ements, does not change during the denaturation process. We interpret these results in terms of the following model: N-6 reversible arrow N- 6' --> 6U(--> F) where N-6 and N-6' are, respectively, the native hexa mer and a hexameric, highly folded high-enthalpy species, U is the unf olded monomer and F is some final aggregated state. The kinetic interm ediate, N-6', possesses the properties of one definition of a molten g lobule, having a very high enthalpy and a hexameric compact structured form, This ''molten globule'' is an obligatory intermediate in the un folding pathway of the protein. The stabilization of the protein by AD P is due to the modulation of the high-enthalpy two-state predenaturat ional E reversible arrow E' transition, resulting in the lowering of t he energy of the native state of the protein.