Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Akane Kawamura, James Graham, Adeel Mushtaq, Stefanos A. Tsiftsoglou, Gregory M. Vath, Patrick E. Hanna, Carston R Wagner, Edith Sim

Research output: Contribution to journalArticle

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Abstract

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Original languageEnglish (US)
Pages (from-to)347-359
Number of pages13
JournalBiochemical Pharmacology
Volume69
Issue number2
DOIs
StatePublished - Jan 15 2005

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Arylamine N-Acetyltransferase
Mesocricetus
Substrate Specificity
Screening
Throughput
Substrates
para-Aminobenzoates
Assays
Hydralazine
Xenobiotics
Enzymes
Eukaryota
Human Activities
Cricetinae
Carcinogens
Pharmaceutical Preparations
N-acetyltalosaminuronic acid
Proteins

Keywords

  • AcetylCoA/CoA derivatives
  • Arylamine N-acetyltransferase
  • Arylamines
  • High-throughput screening
  • Recombinant protein
  • Xenobiotic metabolism

Cite this

Kawamura, A., Graham, J., Mushtaq, A., Tsiftsoglou, S. A., Vath, G. M., Hanna, P. E., ... Sim, E. (2005). Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening. Biochemical Pharmacology, 69(2), 347-359. https://doi.org/10.1016/j.bcp.2004.09.014

Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening. / Kawamura, Akane; Graham, James; Mushtaq, Adeel; Tsiftsoglou, Stefanos A.; Vath, Gregory M.; Hanna, Patrick E.; Wagner, Carston R; Sim, Edith.

In: Biochemical Pharmacology, Vol. 69, No. 2, 15.01.2005, p. 347-359.

Research output: Contribution to journalArticle

Kawamura, Akane ; Graham, James ; Mushtaq, Adeel ; Tsiftsoglou, Stefanos A. ; Vath, Gregory M. ; Hanna, Patrick E. ; Wagner, Carston R ; Sim, Edith. / Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening. In: Biochemical Pharmacology. 2005 ; Vol. 69, No. 2. pp. 347-359.
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