Catalytic Mechanism of NADP+-Dependent Isocitrate Dehydrogenase: Implications from the Structures of Magnesium-Isocitrate and NADP+ Complexes

James H. Hurley, Antony M. Dean, Daniel E. Koshland, Robert M. Stroud

Research output: Contribution to journalArticle

225 Scopus citations

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-Å 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 de¬hydrogenase-like and the lactate dehydrogenase-like classes of nucleotide-binding proteins. In the mag- nesium-isocitrate complex, magnesium is coordinated to the α-carboxylate and α-hydroxyl oxygen of isocitrate in a manner suitable for stabilization of a negative charge on the hydroxyl oxygen during both the dehy¬drogenation and decarboxylation steps of the conversion of isocitrate to α-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 2833′. 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.

Original languageEnglish (US)
Pages (from-to)8671-8678
Number of pages8
JournalBiochemistry
Volume30
Issue number35
DOIs
StatePublished - Sep 1 1991

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