Structural And Functional Studies Of Human Cytosolic NADP-dependent Isocitrate Dehydrogenase

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation ofisocitrate to alpha-ketoglutarate;regulation of the enzymatic activity of IDHs iscrucial for their biological functions. Bacterial IDHs are proved to use a randomsteady-state catalytic mechanism and can be reversibly regulated by phosphorylationof a strictly conserved serine residue at the active site. Eukaryotic NADP-dependentIDHs (NADP-IDHs) have been shown to have several important biological functions;however, their kinetic and regulatory mechanism remains unclear. Structural studiesof human cytosolic NADP-IDH (HcIDH) in complex with various combinations ofsubstrate, cofactor and metal ion reveal three conformational states of the enzyme thatdiffer substantially in overall structure and in the structure of the active site. Astructural segment at the active site forms a conserved alpha-helix in all knownNADP-IDH structures but assumes a loop conformation in the open, inactive form ofHcIDH;partially unraveled alpha-helix in the semi-open, intermediate form;and analpha-helix in the closed, active form. The structural information, taken together withprevious biochemical data led us to propose a self-regulatory mechanism for HcIDHthat mimics the phosphorylation mechanism used by the bacterial homologs.Moreover, a random steady-state kinetic mechanism can be applied to HcIDH. Ourresults also provide insights into substrate and cofactor recognition and specificity byeukaryotic NADP-IDHs, as well as their biological significances.