Isocitrate dehydrogenase (NADP+)

 

Isocitrate dehydrogenase (ICD) is a crucial enzyme in the TCA cycle. Reversible inactivation of the enzyme by phosphorylation at Ser113 regulates metabolic switching from the TCA cycle to the glyoxylate pathway, which is adopted by bacteria, fungi and plants in the absence of a "complex" carbon source such as glucose. In humans, specific mutations within ICD are found in several forms of brain tumour, with some specificity between pathologies. Mutations in ICD are detected in almost all forms of secondary glioblastoma which form from lower-grade glioma, but they are rarely found in primary high-grade glioblastoma multiforme. A correlation between the presence of a ICD mutation and increased life expectancy from secondary glioblastoma has been suggested [PMID:19636000].

 

Reference Protein and Structure

Sequence
P08200 UniProt (1.1.1.42) IPR004439 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
5icd - REGULATION OF AN ENZYME BY PHOSPHORYLATION AT THE ACTIVE SITE (2.5 Å) PDBe PDBsum 5icd
Catalytic CATH Domains
3.40.718.10 CATHdb (see all for 5icd)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.1.1.42)

NADP(+)
CHEBI:18009ChEBI
+
D-threo-isocitrate(3-)
CHEBI:15562ChEBI
→
2-oxoglutarate(2-)
CHEBI:16810ChEBI
+
NADPH
CHEBI:16474ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
Alternative enzyme names: NADP isocitric dehydrogenase, NADP-dependent isocitrate dehydrogenase, NADP-dependent isocitric dehydrogenase, NADP-linked isocitrate dehydrogenase, NADP-specific isocitrate dehydrogenase, NADP(+)-linked isocitrate dehydrogenase, Isocitrate (NADP) dehydrogenase, Isocitrate (nicotinamide adenine dinucleotide phosphate) dehydrogenase, Isocitrate dehydrogenase (NADP), Isocitrate dehydrogenase (NADP-dependent), Oxalosuccinate decarboxylase, Oxalsuccinic decarboxylase, IDH, Triphosphopyridine nucleotide-linked isocitrate dehydrogenase-oxalosuccinate carboxylase, Dual-cofactor-specific isocitrate dehydrogenase, NADP(+)-ICDH, NADP(+)-IDH, IDP, IDP1, IDP2, IDP3,

Enzyme Mechanism

Introduction

Asp283AA activates is hydrogen bonded to both Lys230 and Tyr160. In this mechanism Asp activates the Lys to act as the general base in the first step and the Tyr to be the general acid in the last step. The hydride transfer from substrate to NADP+ is concerted with the initial deprotonation.

The second half of the reaction involves decarboxylation from C2, forming an enolate which is stabilised by the divalent cation and proximal positively charged residues. This intermediate then undergoes a rearrangement to form the final product with Tyr as the general acid.

Catalytic Residues Roles

UniProt PDB* (5icd)
Tyr160 Tyr160A Acts as a general acid/base and donates its proton to the final product. It is returned to its correct protonation state by abstracting a proton from Asp283. hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Asp307 Asp307A Binds Mg(II) ion. metal ligand
Asp283 Asp283A(AA) Acts as the general acid/base, performs the initial deprotonation of Lys230. Reprotonates Tyr160. proton acceptor, electrostatic stabiliser, proton donor
Lys230 Lys230A(AA) Acts as a general acid/base, it is activated for its initial proton transfer by Asp230. It then abstracts a proton from the substrate. proton acceptor, proton relay, electrostatic stabiliser, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, hydride transfer, bimolecular elimination, aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation, unimolecular elimination by the conjugate base, decarboxylation, intermediate collapse, assisted keto-enol tautomerisation, intermediate terminated

References

  1. Neves RPP et al. (2016), ACS Catal, 6, 357-368. Unveiling the Catalytic Mechanism of NADP+-Dependent Isocitrate Dehydrogenase with QM/MM Calculations. DOI:10.1021/acscatal.5b01928.
  2. Dang L et al. (2009), Nature, 462, 739-744. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. DOI:10.1038/nature08617. PMID:19935646.

Step 1. Asp283AA deprotonates Lys230, which deprotonates the C2-OH, which causes the elimination of a hydride ion, which adds to NADP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys230A(AA) electrostatic stabiliser
Asp307A metal ligand
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp283A(AA) proton acceptor
Lys230A(AA) proton donor, proton acceptor, proton relay

Chemical Components

proton transfer, hydride transfer, ingold: bimolecular elimination, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation

Step 2. The intermediate decarboxylates, with concomitant double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Asp307A metal ligand
Tyr160A electrostatic stabiliser
Asp283A(AA) electrostatic stabiliser
Lys230A(AA) electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, decarboxylation, intermediate collapse, intermediate formation

Step 3. The oxyanion formed collapses, with concomitant deprotonation of Tyr160A, in turn re-protonated by Asp238AA

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Asp307A metal ligand
Lys230A(AA) electrostatic stabiliser
Tyr160A proton donor, proton acceptor
Asp283A(AA) proton donor
Tyr160A proton relay

Chemical Components

assisted keto-enol tautomerisation, intermediate terminated, overall product formed, proton transfer
Download: Image, Marvin File

Step 1. Asp283AA deprotonates Lys230, which deprotonates the C2-OH, which causes the elimination of a hydride ion, which adds to NADP.

Step 2. The intermediate decarboxylates, with concomitant double bond rearrangement.

Step 3. The oxyanion formed collapses, with concomitant deprotonation of Tyr160A, in turn re-protonated by Asp238AA

Products.

Introduction

Asp283AA acts as the base towards isocitrate C1-OH, initiating simultaneous hydride transfer to NADP+ while forming a carbonyl at this position. The second half of the reaction involves decarboxylation from C2, forming an enolate which is stabilised by the divalent cation and proximal positively charged residues. This intermediate then undergoes a rearrangement to form the C2-carbonyl and form alpha-ketoglutarate.

Catalytic Residues Roles

UniProt PDB* (5icd)
Tyr160 Tyr160A Activates Asp283 hydrogen bond donor, electrostatic stabiliser
Asp307 Asp307A Binds Mg(II) ion. metal ligand
Asp283 Asp283A(AA) Acts as the general acid/base, performs the initial deprotonation of the substrate. It is subsequently deprotonated by Lys230 in an inferred return step. proton acceptor, electrostatic stabiliser, proton donor
Lys230 Lys230A(AA) Acts as a general acid/base, donating a proton to the final product. It is reprotonated by Asp283. proton acceptor, electrostatic stabiliser, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular elimination, aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation, hydride transfer, proton transfer, unimolecular elimination by the conjugate base, decarboxylation, intermediate collapse, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Hurley JH et al. (1991), Biochemistry, 30, 8671-8678. Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. DOI:10.1021/bi00099a026. PMID:1888729.
  2. Sanson M et al. (2009), J Clin Oncol, 27, 4150-4154. Isocitrate Dehydrogenase 1 Codon 132 Mutation Is an Important Prognostic Biomarker in Gliomas. DOI:10.1200/jco.2009.21.9832. PMID:19636000.
  3. Peng Y et al. (2008), Protein Sci, 17, 1542-1554. Structural studies ofSaccharomyces cerevesiaemitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction. DOI:10.1110/ps.035675.108. PMID:18552125.
  4. Kim TK et al. (2005), Protein Sci, 14, 140-147. Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase. DOI:10.1110/ps.041091805. PMID:15576556.
  5. Doyle SA et al. (2001), Biochemistry, 40, 4234-4241. Structural Basis for a Change in Substrate Specificity:  Crystal Structure of S113E Isocitrate Dehydrogenase in a Complex with Isopropylmalate, Mg2+, and NADP†,‡. DOI:10.1021/bi002533q. PMID:11284679.
  6. Imada K et al. (1998), Structure, 6, 971-982. Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 å resolution: the role of Glu88 in the unique substrate-recognition mechanism. DOI:10.1016/s0969-2126(98)00099-9. PMID:9739088.
  7. Lee ME et al. (1995), Biochemistry, 34, 378-384. Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP+-dependent isocitrate dehydrogenase from Escherichia coli. DOI:10.1021/bi00001a046. PMID:7819221.
  8. Dean AM et al. (1993), Biochemistry, 32, 9302-9309. Kinetic mechanism of Escherichia coli isocitrate dehydrogenase. DOI:10.1021/bi00087a007. PMID:8369299.
  9. Thorsness PE et al. (1987), J Biol Chem, 262, 10422-10425. Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. PMID:3112144.

Step 1. Asp283AA deprotonates the C2-OH, which causes the elimination of a hydride ion, which adds to NADP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp307A metal ligand
Lys230A(AA) electrostatic stabiliser
Asp283A(AA) proton acceptor

Chemical Components

ingold: bimolecular elimination, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation, hydride transfer, proton transfer

Step 2. The intermediate decarboxylates, with concomitant double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Tyr160A electrostatic stabiliser
Asp307A metal ligand
Asp283A(AA) electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, decarboxylation, intermediate collapse, intermediate formation

Step 3. The oxyanion formed collapses, with concomitant deprotonation of Lys230B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp283A(AA) electrostatic stabiliser
Lys230A(AA) proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, overall product formed, intermediate terminated

Step 4. Lys230AA deprotonates Asp283AA in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Asp307A metal ligand
Tyr160A electrostatic stabiliser
Lys230A(AA) proton acceptor
Asp283A(AA) proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp283AA deprotonates the C2-OH, which causes the elimination of a hydride ion, which adds to NADP.

Step 2. The intermediate decarboxylates, with concomitant double bond rearrangement.

Step 3. The oxyanion formed collapses, with concomitant deprotonation of Lys230B.

Step 4. Lys230AA deprotonates Asp283AA in an inferred return step.

Products.

Introduction

Asp283AA acts as the base towards isocitrate C1-OH, the charged intermediate is well stabilised by the Mg(II) ion, the hydride transfer from the intermediate to NADP+, forming a carbonyl at the C1 position, occurs in a separate step. The second half of the reaction involves decarboxylation from C2, forming an enolate which is stabilised by the divalent cation and proximal positively charged residues. This intermediate then undergoes a rearrangement to form the C2-carbonyl and form alpha-ketoglutarate.

Catalytic Residues Roles

UniProt PDB* (5icd)
Tyr160 Tyr160A Helps activate Asp283. hydrogen bond donor, electrostatic stabiliser
Asp307 Asp307A Binds the Mg(II) ion. metal ligand
Asp283 Asp283A(AA) Acts as a general acid/base, performing the initial deprotonation of the substrate. Lys230 deprotonates Asp283 to regenerate the starting state of the active site. proton acceptor, electrostatic stabiliser, proton donor
Lys230 Lys230A(AA) Acts as a general acid/base, reprotonating the product. Lys230 deprotonates Asp283 ro regenerate the starting state of the active site. proton acceptor, electrostatic stabiliser, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall reactant used, intermediate formation, proton transfer, hydride transfer, bimolecular elimination, aromatic bimolecular nucleophilic addition, overall product formed, unimolecular elimination by the conjugate base, decarboxylation, intermediate collapse, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Hurley JH et al. (1991), Biochemistry, 30, 8671-8678. Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. DOI:10.1021/bi00099a026. PMID:1888729.

Step 1. Asp283AA deprotonates the C2-OH, the resulting anionic intermediate is well stabilised by the Mg(II) ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys230A(AA) electrostatic stabiliser
Asp307A metal ligand
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp283A(AA) proton acceptor

Chemical Components

overall reactant used, intermediate formation, proton transfer

Step 2. A hydride ion is eliminated from the intermediate and adds to NADP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys230A(AA) electrostatic stabiliser
Asp307A metal ligand
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp283A(AA) electrostatic stabiliser

Chemical Components

hydride transfer, ingold: bimolecular elimination, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation

Step 3. The intermediate decarboxylates, with concomitant double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Asp307A metal ligand
Asp283A(AA) electrostatic stabiliser
Tyr160A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, decarboxylation, intermediate collapse, intermediate formation

Step 4. The oxyanion formed collapses, with concomitant deprotonation of Lys230B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor, electrostatic stabiliser
Asp283A(AA) electrostatic stabiliser
Lys230A(AA) proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, overall product formed, intermediate terminated

Step 5. Lys230AA deprotonates Asp283AA in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr160A hydrogen bond donor
Asp307A metal ligand
Tyr160A electrostatic stabiliser
Lys230A(AA) proton acceptor
Asp283A(AA) proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp283AA deprotonates the C2-OH, the resulting anionic intermediate is well stabilised by the Mg(II) ion.

Step 2. A hydride ion is eliminated from the intermediate and adds to NADP.

Step 3. The intermediate decarboxylates, with concomitant double bond rearrangement.

Step 4. The oxyanion formed collapses, with concomitant deprotonation of Lys230B.

Step 5. Lys230AA deprotonates Asp283AA in an inferred return step.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Katherine Ferris