Aconitate hydratase

 

Aconitase (aconitate hydratase) contains an Fe-S cluster within the active site. This can be in the form 4Fe-4S in which case the enzyme functions as an aconitate hydratase, performing the dehydration-rehydration of citrate to isocitrate via cis-aconitate in the second and third steps of the Krebs cycle. When the Fe-S cluster is in the form 3Fe-4S (cytosolic aconitase) the enzyme functions as an Iron Regulatory Protein (IRP) which binds to a conserved sequence (Iron Responsive Element; IRE) in mRNAs encoding proteins that function in the maintenance of iron homeostasis thus regulating the translation of that mRNA. Aconitase occurs in bacteria, plants, animals and fungi in mitochondrial and cytosolic forms.

 

Reference Protein and Structure

Sequence
P20004 UniProt (4.2.1.3) IPR006248 (Sequence Homologues) (PDB Homologues)
Biological species
Bos taurus (Cattle) Uniprot
PDB
1fgh - COMPLEX WITH 4-HYDROXY-TRANS-ACONITATE (2.05 Å) PDBe PDBsum 1fgh
Catalytic CATH Domains
3.30.499.10 CATHdb 3.20.19.10 CATHdb (see all for 1fgh)
Cofactors
Water (1), Tetra-mu3-sulfido-tetrairon (1)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.3)

isocitrate(3-)
CHEBI:16087ChEBI
→
citrate(3-)
CHEBI:16947ChEBI
Alternative enzyme names: Cis-aconitase, Aconitase, Citrate hydro-lyase, Citrate(isocitrate) hydro-lyase, AcnB, 2-methylaconitate hydratase,

Enzyme Mechanism

Introduction

Ser642, the general base is thought to be deprotonated when the isocitrate substrate binds to the Fe centre and Fe-OH2 is formed in the coordination sphere. Serine 642 abstracts the proton from the beta-carbon of isocitrate to form cis-aconitate. Conjugate elimination of the hydroxyl group and cleavage of the alpha C-OH bond results in the deprotonation of His101. The His101-Asp100 pair activate the Fe coordinated water molecule to provide a hydroxide to cis-aconitate. Ser642 then protonates the alpha carbon of this intermediate to form citrate.

Catalytic Residues Roles

UniProt PDB* (1fgh)
His128 His101A The residue acts as a general acid to the eliminated hydroxyl from isocitrate. Hydrogen bonding with Asp100 stabilises the residue's positive charge. proton acceptor, electrostatic stabiliser, proton donor
Asp127 Asp100A The residue stabilises the positive charge on His101, facilitating the the role of His101 as a general acid. electrostatic stabiliser, increase acidity
Arg671 (main-N) Arg644A (main-N) The residue's backbone amide stabilises the alkoxide transition state in the elimination of OH from the isocitrate substrate. electrostatic stabiliser
Arg474 Arg447A The interaction between the residue's gaunidinium group and one of the substrate carboxylates enhances the active site specificity for substrates structure and stereochemistry. electrostatic stabiliser
Ser669 Ser642A The residue undergoes rapid proton exchange on binding of the substrate and water to the Fe metal centre. In the ionised form the residue then acts as a general base towards isocitrate, abstracting the beta-carbon proton. After elimination and reinsertion of water, Ser642 acts as a general acid to the alpha carbon of the cis-aconitate intermediate, forming the citrate product. proton acceptor, proton donor
Asp192 Asp165A The residue hydrogen bonds to both the hydroxyl group of the isocitrate and the coordinated hydroxide. This allows Asp165 to orientate the substrate correctly within the metal coordination sphere for catalysis. electrostatic stabiliser
*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, bimolecular elimination, overall reactant used, bimolecular nucleophilic addition, overall product formed, native state of enzyme regenerated

References

  1. Brown NM et al. (1998), Proc Natl Acad Sci U S A, 95, 15235-15240. Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1. DOI:10.1073/pnas.95.26.15235. PMID:9860952.
  2. Lloyd SJ et al. (1999), Protein Sci, 8, 2655-2662. The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex. DOI:10.1110/ps.8.12.2655. PMID:10631981.
  3. Irvin SD et al. (1998), J Mol Evol, 46, 401-408. A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms. DOI:10.1007/pl00006319. PMID:9541534.
  4. Lauble H et al. (1994), J Mol Biol, 237, 437-451. Crystal structures of aconitase with trans-aconitate and nitrocitrate bound. DOI:10.1006/jmbi.1994.1246. PMID:8151704.
  5. Zheng L et al. (1992), J Biol Chem, 267, 7895-7903. Mutational analysis of active site residues in pig heart aconitase. PMID:1313811.
  6. Lauble H et al. (1992), Biochemistry, 31, 2735-2748. Crystal structures of aconitase with isocitrate and nitroisocitrate bound. DOI:10.1021/bi00125a014. PMID:1547214.

Step 1. Ser642 acts as a general base and deprotonates the beta-carbon of isocitrate. This causes the elimination of the alpha carbon hydroxyl upon protonation by His101 and a cis-aconitate intermediate is formed.

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Catalytic Residues Roles

Residue Roles
His101A electrostatic stabiliser
Asp100A electrostatic stabiliser
Asp165A electrostatic stabiliser
Arg447A electrostatic stabiliser
Arg644A (main-N) electrostatic stabiliser
Asp100A increase acidity
His101A proton donor
Ser642A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used

Step 2. His 101 activates the Fe coordinated water which performs a nucleophilic on the beta-carbon. Simultaneously Ser642 acts as a general acid donating a proton to the alpha-carbon, forming the citrate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp100A electrostatic stabiliser
His101A electrostatic stabiliser
Asp165A electrostatic stabiliser
Arg447A electrostatic stabiliser
Arg644A (main-N) electrostatic stabiliser
Ser642A proton donor
His101A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, proton transfer, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Ser642 acts as a general base and deprotonates the beta-carbon of isocitrate. This causes the elimination of the alpha carbon hydroxyl upon protonation by His101 and a cis-aconitate intermediate is formed.

Step 2. His 101 activates the Fe coordinated water which performs a nucleophilic on the beta-carbon. Simultaneously Ser642 acts as a general acid donating a proton to the alpha-carbon, forming the citrate product.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday, James Willey