N-hydroxyarylamine O-acetyltransferase

 

The N acetyl-transferase (NAT) family of enzymes N-acetylate arlyhydrazines and arylamines. The transfer of an acetyl group from acetyl coenzyme A to a substrate was first noted in the deactivation of the drug isonizid, where the rate of deactivation varied between individuals, as explained by pharmacogenetic variation in the rate of NAT activity. NAT enzymes are also involved in the metabolism and detoxification of arylamine and arylhydroxylamine carcinogenic substrates.

 

Reference Protein and Structure

Sequence
Q00267 UniProt (2.3.1.5, 2.3.1.118) IPR001447 (Sequence Homologues) (PDB Homologues)
Biological species
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (Bacteria) Uniprot
PDB
1e2t - Arylamine N-acetyltransferase (NAT) from Salmonella typhimurium (2.8 Å) PDBe PDBsum 1e2t
Catalytic CATH Domains
2.40.128.150 CATHdb 3.30.1120.150 CATHdb (see all for 1e2t)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.118)

acetyl-CoA(4-)
CHEBI:57288ChEBI
+
N-hydroxyarylamine
CHEBI:13792ChEBI
→
coenzyme A(4-)
CHEBI:57287ChEBI
+
N-acetoxyarylamine
CHEBI:21494ChEBI
Alternative enzyme names: N-hydroxy-2-aminofluorene-O-acetyltransferase, Arylamine N-acetyltransferase, Arylhydroxamate N,O-acetyltransferase,

Enzyme Mechanism

Introduction

The N-acetylation mechanism involves the transfer of an acetyl group from acetyl coenzyme A to the aryl substrate. The main catalytic residue, Cys 69 has been shown to form a reactive acyl-enzyme intermediate which is readily deacetylated by the basic N atom of the amine substrate. The close proximity Arg 64, along with the conserved Glu 39 are thought to stabilise the conformation of the Cys 69, and are not involved in a general base activation mechanism at Cys 69 as has been suggested in some work. The low pKa of Cys 69 associated with the formation of a nucleophilic thiolate at physiological pH levels results in a highly activated acetyl- enzyme intermediate (the lower the pKa, the more stable the conjugate acid).

Catalytic Residues Roles

UniProt PDB* (1e2t)
His107 His107(110)A Forms part of the Cys-His-Asp ctalytic triad. Histidine is responsible for the activation of the catalytic cysteine. modifies pKa, proton shuttle (general acid/base)
Asp122 Asp122(125)A Forms part of the Cys-His-Asp ctalytic triad. The aspartate residue is responsible for activating the histidine of the triad. modifies pKa
Glu39 Glu39(42)A The residue acts to stabilise the conformation of the catalytic Cys 69 through the salt bridge formed with Arg 64, which is close in proximity to the catalytic residue. electrostatic stabiliser
Arg64 Arg64(67)A The residue acts to stabilise the conformation of the catalytic Cys 69 through the salt bridge formed with the conserved Glu 39 residue. The residue is remote in space from the catalytic S atom of Cys 69 and so does not act as a general base to activate nucleophilic attack. electrostatic stabiliser
Cys69 Cys69(72)A The catalytic Cys 69 exists as a thiolate at physiological pH level. It acts as a nucleophile towards the acetyl coenzyme A initially forming an acetyl-enzyme intermediate. The low pKa of the thiol group increases the activity of the associated thioester, which increases its susceptibility towards nucleophilic attack of the arylamine or arylhydroxylamine substrate. covalent catalysis, proton shuttle (general acid/base), 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

References

  1. Sinclair JC et al. (2000), Nat Struct Biol, 7, 560-564. Structure of arylamine N-acetyltransferase reveals a catalytic triad. DOI:10.1038/76783. PMID:10876241.
  2. Watanabe M et al. (1994), Environ Health Perspect, 102 Suppl 6, 83-89. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes. PMID:7889864.
  3. Watanabe M et al. (1992), J Biol Chem, 267, 8429-8436. Involvement of Cys69 residue in the catalytic mechanism of N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium. Sequence similarity at the amino acid level suggests a common catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms. PMID:1569093.
  4. Andres HH et al. (1988), J Biol Chem, 263, 7521-7527. On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J). PMID:2897358.

Step 1.

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Catalytic Residues Roles

Residue Roles
Glu39(42)A electrostatic stabiliser
Cys69(72)A electrostatic stabiliser
Arg64(67)A electrostatic stabiliser
Cys69(72)A proton shuttle (general acid/base)
His107(110)A proton shuttle (general acid/base)
Asp122(125)A modifies pKa
Cys69(72)A covalent catalysis
His107(110)A modifies pKa

Chemical Components

Step 1.

Contributors

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