Phosphomimetic mutations near active sites of proteins in Thermus thermophilus suggest a widespread regulatory mechanism.

In Thermus thermophilus, an aerobic Gram-negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand-binding site, including the active site, of the enzyme in the three-dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild-type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.