Monovalent cation induced reassociation of formyltetrahydrofolate synthetase monitored by Rayleigh light scattering and enzymic activity.

It has been previously established that formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum is reversibly dissociated and inactivated in the absence of certain monovalent cations. In the present paper, the reassociation of monomeric, inactive enzyme to form tetrameric, active enzyme was monitored by Rayleigh light scattering and enzymic activity. Light-scattering measurements confirmed that the active enzyme is composed of four subunits of equal weight. With the assumption that the results of analytical ultracentrifugation are correct--that monomers and tetramers are the only species ever present at appreciable levels--the amount of tetramer formed during reassociation was calculated from the light-scattering data. Evidence for the accumulation of catalytically active intermediates was obtained by comparing the rate of association of monomers (detected by light scattering) to the rate of return of enzymic activity. The accumulation of intermediates was most strikingly seen at low monovalent cation concentration at low ionic strength. Evidence is also presented that sedimentation favors reassociation of the enzyme. The reassociation data were fit to a second-order reversible rate equation. Interestingly, although the data were derived from the same experiments, the kinetic plot based on light-scattering measurements yielded a straight line function with an abrupt change in slope about 10 min after initiation of reassociation, while plots based on enzymic activity measurements gave a single slope.