Fumarylacetoacetase

 

Fumarylacetoacetate hydrolase (FAH) catalyses hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to give fumarate and acetoacetate - this step occurs as the final step in Phe and Tyr degradation. FAH functions as a metalloenzyme.

Herditary tyrosinemia type I is a fatal disease caused by loss of FAH activity. This enzyme also catalyses reactions required by soil bacteria to degrade aromatic hydrocarbons, and so understanding of this enzyme may have possible applications in bioengineering efforts to bioremediate toxic hydrocarbon wastes.

 

Reference Protein and Structure

Sequence
P35505 UniProt (3.7.1.2) IPR005959 (Sequence Homologues) (PDB Homologues)
Biological species
Mus musculus (house mouse) Uniprot
PDB
1hyo - CRYSTAL STRUCTURE OF FUMARYLACETOACETATE HYDROLASE COMPLEXED WITH 4-(HYDROXYMETHYLPHOSPHINOYL)-3-OXO-BUTANOIC ACID (1.3 Å) PDBe PDBsum 1hyo
Catalytic CATH Domains
3.90.850.10 CATHdb (see all for 1hyo)
Cofactors
Magnesium(2+) (1), Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.7.1.2)

4-fumarylacetoacetate(2-)
CHEBI:18034ChEBI
+
water
CHEBI:15377ChEBI
→
acetoacetate
CHEBI:13705ChEBI
+
fumarate(2-)
CHEBI:29806ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Beta-diketonase, Fumarylacetoacetate hydrolase,

Enzyme Mechanism

Introduction

The catalytic mechanism of fumarylacetoacetate hydrolase begins with binding of the fumarylacetoacetate substrate, which is activated by the Ca ion. A nucleophilic water molecule is activated by the His 133 and Glu 199, which is itself primed by the calcium ion, and it attacks the substrate facilitated by proton abstraction by His 133, which is being activated in turn by Glu 364. The formation of a tetrahedral alkoxide transition state is stabilised by the oxyanion hole formed by Gln 240, Lys 253 and Arg 237 as well as the Ca ion. Collapse of the transition state occurs with proton donation by Lys 253, with formation of the acetoacetate leaving group stabilised by the Ca ion.

Catalytic Residues Roles

UniProt PDB* (1hyo)
Lys253 (main-C), Thr257 Lys253(255)A (main-C), Thr257(259)A Forms part of the magnesium binding site. metal ligand
Glu364 Glu364(366)A Activates His 133 as part of the His-Glu dyad. hydrogen bond acceptor, electrostatic stabiliser
Glu199 Glu199(201)A Activates water for nucleophilic attack. Also forms part of the calcium binding site. hydrogen bond acceptor, metal ligand, steric role
Asp233 Asp233(235)A Acts as a bridging ligand between the two metal ions. metal ligand
Arg237, Gln240 Arg237(239)A, Gln240(242)A Forms part of the oxyanion hole. hydrogen bond donor, electrostatic stabiliser
Lys253 Lys253(255)A Forms part of the oxyanion hole, and donates a proton as a general acid catalyst in the collapse of the tetrahedral transition state. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
His133 His133(135)A Activates water by proton abstraction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp126, Glu201 Asp126(128)A, Glu201(203)A Forms part of the calcium binding site. metal ligand
*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 nucleophilic addition, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, intermediate terminated, native state of enzyme regenerated

References

  1. Bateman RL et al. (2001), J Biol Chem, 276, 15284-15291. Mechanistic Inferences from the Crystal Structure of Fumarylacetoacetate Hydrolase with a Bound Phosphorus-based Inhibitor. DOI:10.1074/jbc.m007621200. PMID:11154690.
  2. Ran T et al. (2013), Biochem J, 449, 51-60. Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family. DOI:10.1042/BJ20120913. PMID:23046410.
  3. Timm DE et al. (1999), Structure, 7, 1023-1033. Crystal structure and mechanism of a carbon–carbon bond hydrolase. DOI:10.1016/s0969-2126(99)80170-1. PMID:10508789.

Step 1. His133 deprotonates water, which attacks the carbonyl carbon in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg237(239)A hydrogen bond donor, electrostatic stabiliser
Gln240(242)A hydrogen bond donor, electrostatic stabiliser
Lys253(255)A hydrogen bond donor, electrostatic stabiliser
Glu199(201)A metal ligand, steric role, hydrogen bond acceptor
His133(135)A hydrogen bond donor, hydrogen bond acceptor
Glu364(366)A hydrogen bond acceptor
Asp233(235)A metal ligand
Thr257(259)A metal ligand
Asp126(128)A metal ligand
Glu201(203)A metal ligand
Lys253(255)A (main-C) metal ligand
His133(135)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 2. The oxyanion initiates an elimination that cleaves the C-C bond, resulting in a carbanion and the fumarate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg237(239)A hydrogen bond donor, electrostatic stabiliser
Gln240(242)A hydrogen bond donor, electrostatic stabiliser
Lys253(255)A hydrogen bond donor, electrostatic stabiliser
Glu199(201)A metal ligand
His133(135)A hydrogen bond donor
Glu364(366)A hydrogen bond acceptor, electrostatic stabiliser
Asp233(235)A metal ligand
Thr257(259)A metal ligand
Asp126(128)A metal ligand
Glu201(203)A metal ligand

Chemical Components

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

Step 3. The carbanion deprotonates Lys253, forming the acetoacetate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg237(239)A hydrogen bond donor
Gln240(242)A hydrogen bond donor
Lys253(255)A hydrogen bond donor
Glu199(201)A metal ligand
His133(135)A hydrogen bond donor
Glu364(366)A hydrogen bond acceptor, electrostatic stabiliser
Asp233(235)A metal ligand
Thr257(259)A metal ligand
Asp126(128)A metal ligand
Glu201(203)A metal ligand
Lys253(255)A proton donor

Chemical Components

proton transfer, intermediate terminated, overall product formed

Step 4. Water deprotonates His133 and Lys253 deprotonates water to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys253(255)A hydrogen bond acceptor
Glu199(201)A metal ligand
His133(135)A hydrogen bond donor
Glu364(366)A hydrogen bond acceptor, electrostatic stabiliser
Asp233(235)A metal ligand
Thr257(259)A metal ligand
Asp126(128)A metal ligand
Glu201(203)A metal ligand
His133(135)A proton donor
Lys253(255)A proton acceptor

Chemical Components

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

Step 1. His133 deprotonates water, which attacks the carbonyl carbon in a nucleophilic addition.

Step 2. The oxyanion initiates an elimination that cleaves the C-C bond, resulting in a carbanion and the fumarate product.

Step 3. The carbanion deprotonates Lys253, forming the acetoacetate product.

Step 4. Water deprotonates His133 and Lys253 deprotonates water to regenerate the active site.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gary McDowell, Charity Hornby