Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bif unctional protein, IDH kinase/phosphatase, In this paper, we demonstrate th at the effecters controlling these activities belong to two distinct classe s that differ in mechanism and in the locations of their binding sites. NAD PH and isocitrate are representative members of one of these effector class es. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase, Isocitrate can "activate" IDH phosphatase by reve rsing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that t hese ligands regulate IDH kinase/phosphatase through the IDH active site. K inetic analyses suggested that isocitrate and NADPH prevent formation of th e complex between IDH kinase/phosphatase and its protein substrate. AMP, 3- phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands b ind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of th is enzyme. These effector classes can also be distinguished by the observat ion that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphogl ycerate, and pyruvate but not to NADPH and isocitrate.