HETERODIMERIC THYMIDYLATE SYNTHESES WITH C-TERMINAL DELETION ON ONE SUBUNIT

We have combined site directed mutagenesis with the technique of rever sible unfolding and subunit dissociation to construct heterodimeric th ymidylate syntheses that lack the C-terminal valine from only one subu nit of the dimer. Removal of this residue either from both subunits of the dimer by mutagenesis (V316Am mutation) or from only one subunit b y treatment with carboxypeptidase has been reported to result in an in active enzyme (Carreras, C. W, Climie, S. C., and Santi, D. V. (1992) Biochemistry 31, 6038-6044; Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). Arg-178 is an essential act ive site residue of thymidylate synthese that is donated from the oppo sing subunit of the dimer. The R178F-V316Am heterodimer was formed by the unfolding and refolding of a mixture of inactive R178F and V316Am mutants. This enzyme has one intact active site and was found to have half of the activity and the same K-m values as wild-type thymidylate synthase that was unfolded and refolded as a control. We have also for med the V316Am-WT heterodimer and report that this heterodimeric enzym e is also active, has a k(cat) value that is approximately half of tha t of the wild-type thymidylate synthase dimer, and binds substrate and cofactor with K-m values similar to those of the wild-type enzyme.