Inhibitor Binding in the Transition State for Unfolding of Adenosine Deaminase

Inhibitors protect calf intestinal adenosine deaminase from reversible denaturation by guanidine hydrochloride, and from heat inactivation. In the presence of saturating concentrations of ligands, the first-order rate constant for reversible denaturation of this enzyme in 3.0 M guanidine hydrochloride is reduced 6-fold by pteridine or 6-dimethylaminopurine ribonucleoside, and 60-fold by purine ribonucleoside; these compounds provide similar protection against thermal inactivation at 70°C. These protective effects indicate that in the transition state for denaturation by heat or guanidine hydrochloride, the enzyme retains much of its native structure, exhibiting 60-70% of the free energy of association with these ligands that was present in the native enzyme in the ground state. However, neither these ligands nor the extremely powerful inhibitor 2′-deoxycoformycin affect the rate of refolding of enzyme denatured in guanidine hydrochloride.