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.

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

“…Oxalosuccinate will be produced if HtICDH catalyzes only dehydrogenation and if the following decarboxylation is a nonenzymatic reaction. This is an extraordinary idea since it is well established that, for EcICDH, the dehydrogenation and decarboxylation reactions are closely coupled (10). In the case of NADP-dependent ICDH from pig heart, the hypothetical intermediate, oxalosuccinate, is not released from the enzyme (20).…”

“…A preliminary three-dimensional comparison study using a homology modeling tool suggests that the position of D284 in HtICDH has widely diverged from that of the corresponding residue (D283) in EcICDH. Since D283 is one of the substrate binding residues in EcICDH (10), this divergence may be the cause of the low substrate affinity of HtICDH. Further molecular dynamics, crystallographic, and mutagenesis studies are required.…”

The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase

“…IC). A rigidly con- Hurley et al, 1990). These effects are mimicked when SI 13 is served glutamic acid at site 87 in IMDH occupies a position sim-replaced by aspartate or glutamate (Dean et al, 1989; Dean & ilar to SI 13 in IDH.…”

Role of the divalent metal ion in the NAD:malic enzyme reaction: An ESEEM determination of the ground state conformation of malate in the E:Mn: Malate complex

“…This distance range may be compared with the distances between isocitrate and the divalent metal ion determined crystallographically in complexes with isocitrate dehydrogenase. In the Ca-isocitrate-ICDH complex, the distance between Ca and H2 is 4.1 A (Stoddard et al, 1993), and in the Mg-isocitrate-ICDH complex, the Mg-H2 distance is 3.4 A (Hurley et al, 1991). Therefore, the distances derived from the conformational search of Mn-malate complexes appear to be reasonable based on the precedents from analogous complexes defined crystallographically.…”

“…Modeling studies of active site complexes of isopropylmalate dehydrogenase, which also catalyzes a metal-dependent oxidative decarboxylation, led to the proposal that isopropylmalate also binds in the trans conformation (Zhang & Koshland, 1995). In contrast, complexes of isocitrate dehydrogenase with isocitrate show that isocitrate is bound in such a manner that, after oxidation, it will be oriented properly for decarboxylation (Hurley et al, 1991;Stoddard et al, 1993). Interestingly, it has been proposed that isopropylmalate dehydrogenase undergoes a conformational change after binding isopropylmalate (Zhang & Koshland, 1995), whereas no analogous substrate-induced conformational change is believed to occur with isocitrate dehydrogenase (Stoddard et al, 1993).…”