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 bi functional enzyme responsible for the reversible phosphorylation of isocitr ate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the seri ne/threonine protein kinase family. However, only a very limited homology w ith the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino aci ds including three putative residues involved in the kinase activity of IDH K/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterp art eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-direc ted mutagenesis was performed on these three IDHK/P residues, and also on t he 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 purificat ion. Nevertheless, the recovered mutant enzymes were barely able to phospho rylate 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 r esidue altered neither the yield of purification nor the maximal phosphoryl ating capacity of the enzyme. However, when IDH was phosphorylated in the p resence of increasing concentrations of magnesium ions, the two former muta nts 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 essen tially unaffected. Therefore, and in contrast to the current opinion, our r esults suggest that the catalytic mechanism of IDHK/P exhibits some similar ities with that found in the eukaryotic members of the protein kinase famil y.