Hydrogen ion-induced reversible inactivation and dissociation of formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase undergoes inactivation at pH values below 7. Inactivation is accompanied by both the dissociation of the tetramer into monomers and the exposure of aromatic amino acids to the solvent. The rate of inactivation is a function of pH. This rate and the rate of exposure of tryptophan residues to the aqueous environment follow first order kinetics. After prolonged incubation under acidic conditions the monomers are still capable of reassociating to form tetramers but the tetramers produced have a reduced catalytic activity. The alterations in enzyme structure and function which occur during inactivation are reversed following an elevation of pH. The return of enzymic activity is accompanied by both the reassociation of monomers and the burying of exposed tryptophan residues. The rate of reactivation and the rate of burying of tryptophan follow first order kinetics. The first order rate constant for reactivation varies as a function of temperature, and the heat of activation calculated from the linear Arrhenius plot is 20.1 kcal per mole. The free energy of activation and entropy of activation at 25” have values of 21 kcal per mole and -3 e.u., respectively. A mechanism in which the rate-limiting step for reactivation is a conformational change of the monomer that occurs prior to reassociation is consistent with the experimental observations. Both the rate and extent of reactivation increase as the pH is raised from 6 to 7.9. The data suggest that reactivation is dependent on the deprotonation of histidine residues. The presence of an optimum concentration of sulfate enhances both the rate and extent of reactivation at pH values between 6 and 7. The presence of a thiol compound during inactivation and reactivation is required to achieve a maximum restoration of enzymic activity. It is suggested that the monomer is unstable in the absence of a thiol and is converted to other monomer forms possibly as a result of oxidation of sulfhydryl groups. These monomer forms are not converted to the active tetramer under the conditions used.