Three-state kinetic analysis of Chinese hamster dihydrofolate reductase unfolding by guanidine hydrochloride.

The unfolding behavior of dihydrofolate reductase from Chinese hamster in solutions of guanidine hydrochloride (GdnHCl) was studied. The GdnHCl-induced unfolding of the dihydrofolate reductase monitored by intrinsic fluorescence shows a biphasic transition, while the change in the enzyme activity is a single exponential process. The rate constant of inactivation is consistent with that of the fast conformational change. Therefore, the kinetic intermediate of protein unfolding should be a partially folded and inactive form. On the basis of the kinetic equation of substrate reaction in the presence of GdnHCl, all microscopic kinetic constants for the free enzyme and enzyme-substrate complexes have been determined. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation.