Enzyme Memory

Abstract

When a temperature‐jump from 2 to 35°C is applied to wheat‐germ hexokinase LI in solution, the enzyme undergoes an extremely slow conformation change which can be followed by monitoring the fluorescence emission of tryptophanyl residues of the protein. This process occurs in one detectable step and is not associated with any inactivation of the enzyme, although it involves a limited unfolding of the polypeptide chain. The reciprocal of the relaxation time of this slow conformation change (λ 2 ) slightly increases with glucose concentration. A plot of λ 2 against glucose concentration exhibits a downward curvature. If gucose 6‐phosphate is present in the reaction mixture, the reciprocal of the relaxation time, λ 2 , decreases when the ligand concentration is increased. The decay of λ 2 with respect to glucose 6‐phosphate is hyperbolic. This kinetic behavior is identical to that expected for the conformational transition involved in enzyme memory. From this kinetic study one gets the values of the rate constants governing the conformational transition of the enzyme. The forward rate constant k 5′ of the interconversion of the ‘rhombu’ conformation (the conformation state that binds glucose 6‐phosphate) to the ‘circle’ conformation (the conformation state that binds glucose prefenetially) has a value of 10 −3 s −1 , while the backward rate constant k −5′ has a value of about 5 × 10 −6 s −1 . The conformation change is thus almost irreversible; at 35°C and in the absence of any ligand, 99.5% of the free enzyme is under the ‘circle’ conformation. A thermodynamic study of the conversion of the ‘rhombus’ to the ‘circle’ conformation has shown that the entropy of activation Δ S ≠ of this process is highly negative (−42 cal or −175 J K −1 mol −1 ). This is taken to mean that the transition state which has to be reached during the conformation change must have a highly folded structure. This situation clearly distinguish the conformational transition of enzyme memory from a thermal denaturation process.