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.

Abstract	Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) 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.
Authors	Neville J Butcher, Ajanthy Arulpragasam, Rodney F Minchin
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 21 Pg. 22131-7 (May 21 2004) ISSN: 0021-9258 [Print] United States
PMID	15039438 (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
Topics	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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