The subunit structure of formyltetrahydrofolate synthetase

Abstract

The molecular weight of formyltetrahydrofolate synthetase determined by the approach to equilibrium method is 2.38 · 10 5 ± 0.08 · 10 5. In the presence of 1.5–6.0 M guanidine · hydrochloride the enzyme dissociates into subunits. In 6 M guanidine · hydrochloride the molecular weight is 5.98 · 10 4–6.1 · 10 4. The enzyme is therefore a tetramer. Dissociation of the enzyme into monomers also occurs at pH 6, pH 11, and in the absence of certain monovalent cations and multivalent anions. Results of gel filtration experiments done in the presence of the substrates of the reaction and at dilute protein concentrations show that the tetramer is the catalytically active form of the enzyme. Inactivation of the enzyme by dissociation in Tris-HCl is prevented by certain monovalent cations. The presence of 2-mercaptoethanol slows down the inactivation process. The inactivation and dissociation in Tris-HCl containing 2-mercaptoethanol could be reversed by the addition of NH 4Cl. Difference spectroscopy showed that upon reactivation there is an increase in the absorption spectrum of the enzyme in the region where tryptophan, tyrosine and phenylalanine absorb, indicating that some of these residues become buried in a hydrophobic region when the subunits reassociate. The rate of increase in A 291 nm is equal to the rate of return of enzymic activity. NH 4 +, K + and Rb + stimulate the activity of the enzyme 2- to 3-fold under conditions where dissociation does not occur. A plot of the activity as a function of NH 4 + concentration is sigmoidal. It is suggested that the monovalent cations and multivalent anions are necessary to maintain the protein in the most active tetrameric conformation. In the absence of the cation but in the presence of multivalent anions a tetramer of a different conformation is formed. This tetramer has a higher K m for formate. When both cations and multivalent anions are removed the tetramer dissociates into monomers.