Formyltetrahydrofolate Synthetase

Formyltetrahydrofolate synthetase, a tetrameric protein, contains 23 half-cystine residues as determined by cysteic acid analysis and 24 free sulfhydryl groups as determined by four different methods thus indicating absence of disulfide bonds. All of the cysteine residues react with sulfhydryl reagents in the absence of denaturing agents. Titration of the enzyme with p-chloromercuribenzoate (CMB) caused a loss in activity which was a linear function of the amount of thiol groups reacted. A complete loss in activity occurred after 11 CMB molecules had reacted per mole of enzyme. The loss in activity was accompanied by the appearance of the monomeric form of the protein; however, the presence of tetramer which contained CMB was demonstrated by Sephadex G-200 chromatography of enzyme which had been partially inactivated with 14C-labeled CMB. The reaction of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was different from that with CMB. The relation between thiol groups reacted and loss in activity was not linear, and about 18 to 20 thiol groups had to react before a complete loss in activity occurred. Dissociation to the monomeric form did not occur until about 75% of the cysteine residues had reacted. Titration with HgCl2 showed that a linear decrease in activity occurred until complete inactivation was observed after 8 Hg2+ ions had been bound by the protein. Hg2+ could be bound to virtually all of the cysteine residues without causing dissociation of the enzyme. Reactivation by cysteine of enzyme inactivated by CMB or DTNB was variable and could be achieved only when the enzyme was still in the tetrameric form. Total reactivation of mercury enzyme was achieved with 2-mercaptoethanol or cysteine. The inactivation by CMB and DTNB was retarded by ATP and tetrahydrofolate and facilitated by MgATP. Formate and Mg2+ had no effect on the inactivation. It is concluded that the inactivation of formyltetrahydrofolate synthetase by sulfhydryl reagents results from conformational changes produced in the tetramer as well as from dissociation of the tetramer to the inactive monomer. The effects produced by the substrates probably result from conformational changes in the protein. There is no evidence which suggests that a sulfhydryl group or groups constitute part of the active site of the enzyme.