In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher K m for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.The biochemistry of de novo purine nucleotide biosynthesis and salvage is a broadly conserved and well-understood component of cellular metabolism. The first nucleotide formed in the purine biosynthetic pathway of Salmonella enterica is IMP. The synthesis of GTP from IMP involves four steps, with the intermediate formation of GMP and GDP. The enzyme guanylate kinase (Gmk) is a component of this pathway, catalyzing the transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP (or dGMP) ( Fig. 1).As an essential enzyme in the synthesis and salvage of guanine nucleotides, guanylate kinase has been the subject of numerous investigations. In addition, the biomedical role of human Gmk in the activation of antiviral agents has attracted significant attention (2, 13, 14). As a result, a large amount of information has amassed about the biochemistry, genetics, and regulation of these enzymes for a variety of organisms (2,5,6,8,12,13,15,17,20,23) and a high-resolution structure for the Saccharomyces cerevisiae enzyme (GUK1) is available (21). Most guanylate kinases have significant sequence similarity, and the conserved motifs allow the prediction of substrate binding sites. Near the N terminus of the proteins is a canonical nucleoside triphosphate binding motif, GXXXXGK, reflecting the phosphate binding loop (P-loop) that is present in many ATP/GTP binding proteins. This site has been assigned to ATP binding, and the GMP binding site was shown to contain additional conserved residues just C terminal to the P-loop (21).Since the activity of Gmk is essential for growth (e.g., null mutations are lethal), few studies have probed the consequences of altered guanylate kinase activity for cellular metabolism. Mutations in the GUK1 gene of S. cerevisiae were identified for their ability to bypass the purine biosynthetic gene repression normally caused by exogenous adenine (14). In this study, multiple metabolic phenotypes were described for strains that had a detectable, but Ͼ10-fold-decreased, level of GUK activity. The authors attributed the phenotypes of these mutants to the accumulation of the substrate GMP that resulted from the decrease in GUK activity (14). The molecular nature of the causative mutations was not explored in the yeast study, and potential kinetic differences in the mutant enzymes were not addre...