Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: Further evidence that thymidylate synthase is a half-of-the-sites activity enzyme

In a previous study we demonstrated that Escherichia coli thymidylate synth ase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R126E, another inactive mut ant [Maley, F., Pedersen-Lane, J,, and Changchien, L.-M. (1995) Biochemistr y 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a f unctional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was p roposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthas e activity, Y(94)A and R126E The parameters that were examined included p(H ), concentration of protein, temperature, and urea concentration, all of wh ich influenced the rate of activity restoration. It was found, surprisingly , that bp maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly e nhanced, increasing the pH from 6.0 to 9.0 markedly increased the rate at w hich the optimal activity was restored, as did increasing the temperature f rom 4 to 40 degreesC. A similar effect was obtained when the incubation of the mutants was conducted at 4 degreesC in the presence of 1.5 M urea, a te mperature at which activity is restored extremely slowly. Raising the DH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degre esC. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene gly col, and glycerol suggests that changes in subunit interaction and enzyme c onformation are in part responsible for the observed differences. Most sign ificantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitor y. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombi nation to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis, Sequence analysis of the isolated homo- and heterodimers c learly demonstrated that the active enzyme is a product of subunit exchange , one that is very efficient relative to the wild-type enzyme, which did no t exchange subunits unless denatured.