Abstract
A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.
Highlights
A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability
The C-terminal part forms a typical kinase scaffold responsible for the functions of kinase, phosphatase and ATPase, which binds to adenosine triphosphate (ATP) or adenosine diphosphate (ADP)
The KD of AceK is further divided into two lobes: the N-terminal lobe (N-lobe) predominantly forms β-sheet and the larger C-terminal lobe (C-lobe) mainly contains α-helices
Summary
A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Recent theoretical studies showed that the presence of a single Mg2+ ion would lead to a higher energy barrier pathway in the phosphatase reaction than the double Mg2+ ions model, whereas the single metal model would lead to a lower energy barrier pathway in the kinase reaction[28,29] This observation suggests that the metal ion in the catalytic center may have profound but opposing effects on kinase and phosphatase activities. Our study reveals the catalytic role of binuclear metal centers, which helps to understand the reversible reaction catalyzed by the shared active site as well as the regulation of a delicate balance between the kinase and phosphatase activities of AceK
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