title: Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes.
abstract: The structures of NADP+ and magnesium isocitrate bound to the NADP(+)-dependent isocitrate dehydrogenase of Escherichia coli have been determined and refined at 2.5-A resolution. NADP+ is bound by the large domain of isocitrate dehydrogenase, a structure that has little similarity to the supersecondary structure of the nucleotide-binding domain of the lactate dehydrogenase-like family of nucleotide-binding proteins. The coenzyme-binding site confirms the fundamentally different evolution of the isocitrate dehydrogenase-like and the lactate dehydrogenase-like classes of nucleotide-binding proteins. In the magnesium-isocitrate complex, magnesium is coordinated to the alpha-carboxylate and alpha-hydroxyl oxygen of isocitrate in a manner suitable for stabilization of a negative charge on the hydroxyl oxygen during both the dehydrogenation and decarboxylation steps of the conversion of isocitrate to alpha-ketoglutarate. The metal ion is also coordinated by aspartate side chains 283' (of the second subunit of the dimer) and 307 and two water molecules in a roughly octahedral arrangement. On the basis of the geometry of the active site, the base functioning in the dehydrogenation step is most likely aspartate 283'. E. coli isocitrate dehydrogenase transfers a hydride stereospecifically to the A-side of NADP+, and models for a reactive ternary complex consistent with this stereospecificity are discussed.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: J H
authors.initials: JH
authors.lastname: Hurley
authors.forename: A M
authors.initials: AM
authors.lastname: Dean
authors.forename: D E
authors.initials: DE
authors.lastname: Koshland
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D001665:Binding Sites; D007521:Isocitrate Dehydrogenase / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D008274:Magnesium