Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine: a mechanism for the slow acetylator phenotype and substrate-dependent down-regulation.

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives ( approximately 4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.
AuthorsNeville J Butcher, Ajanthy Arulpragasam, Rodney F Minchin
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 21 Pg. 22131-7 (May 21 2004) ISSN: 0021-9258 [Print] United States
PMID15039438 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Cysteine Proteinase Inhibitors
  • DNA, Complementary
  • Isoenzymes
  • Leupeptins
  • Multienzyme Complexes
  • Protein Synthesis Inhibitors
  • Recombinant Proteins
  • Ubiquitin
  • Cycloheximide
  • Arylamine N-Acetyltransferase
  • N-acetyltransferase 1
  • Glutathione Transferase
  • Peptide Hydrolases
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease
  • Cysteine
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde
  • 4-Aminobenzoic Acid
  • 4-Aminobenzoic Acid (pharmacology)
  • Alleles
  • Arylamine N-Acetyltransferase (metabolism)
  • Binding Sites
  • Blotting, Western
  • Cell Line, Tumor
  • Cloning, Molecular
  • Cycloheximide (pharmacology)
  • Cysteine (chemistry)
  • Cysteine Endopeptidases (metabolism)
  • Cysteine Proteinase Inhibitors (pharmacology)
  • DNA, Complementary (metabolism)
  • Down-Regulation
  • Escherichia coli (metabolism)
  • Genotype
  • Glutathione Transferase (metabolism)
  • Humans
  • Isoenzymes (metabolism)
  • Leukocytes, Mononuclear (metabolism)
  • Leupeptins (pharmacology)
  • Models, Biological
  • Multienzyme Complexes (metabolism)
  • Mutagenesis, Site-Directed
  • Mutation
  • Oxidative Stress
  • Peptide Hydrolases (chemistry)
  • Phenotype
  • Precipitin Tests
  • Proteasome Endopeptidase Complex
  • Protein Binding
  • Protein Synthesis Inhibitors (pharmacology)
  • Recombinant Proteins (metabolism)
  • Time Factors
  • Ubiquitin (metabolism)

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