Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII. Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951). In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b). With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press). To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84. Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability. The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.