The “Catalytic” Triad of Isocitrate Dehydrogenase Kinase/Phosphatase from E. coli and Its Relationship with That Found in Eukaryotic Protein Kinases

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK ^- mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K_m value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.