Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):207–215. doi: 10.1042/bj3230207

Study of the role of the highly conserved residues Arg9 and Arg64 in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis.

C Deloménie 1, G H Goodfellow 1, R Krishnamoorthy 1, D M Grant 1, J M Dupret 1
PMCID: PMC1218296  PMID: 9173883

Abstract

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.

Full Text

The Full Text of this article is available as a PDF (743.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andres H. H., Klem A. J., Schopfer L. M., Harrison J. K., Weber W. W. On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J). J Biol Chem. 1988 Jun 5;263(16):7521–7527. [PubMed] [Google Scholar]
  2. Andres H. H., Kolb H. J., Schreiber R. J., Weiss L. Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver. Biochim Biophys Acta. 1983 Aug 16;746(3):193–201. doi: 10.1016/0167-4838(83)90074-2. [DOI] [PubMed] [Google Scholar]
  3. Bell D. A., Taylor J. A., Butler M. A., Stephens E. A., Wiest J., Brubaker L. H., Kadlubar F. F., Lucier G. W. Genotype/phenotype discordance for human arylamine N-acetyltransferase (NAT2) reveals a new slow-acetylator allele common in African-Americans. Carcinogenesis. 1993 Aug;14(8):1689–1692. doi: 10.1093/carcin/14.8.1689. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Cheon H. G., Hanna P. E. Effect of group-selective modification reagents on arylamine N-acetyltransferase activities. Biochem Pharmacol. 1992 May 28;43(10):2255–2268. doi: 10.1016/0006-2952(92)90185-l. [DOI] [PubMed] [Google Scholar]
  6. Deloménie C., Sica L., Grant D. M., Krishnamoorthy R., Dupret J. M. Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations. Pharmacogenetics. 1996 Apr;6(2):177–185. doi: 10.1097/00008571-199604000-00004. [DOI] [PubMed] [Google Scholar]
  7. Dupret J. M., Goodfellow G. H., Janezic S. A., Grant D. M. Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli. J Biol Chem. 1994 Oct 28;269(43):26830–26835. [PubMed] [Google Scholar]
  8. Dupret J. M., Grant D. M. Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2. J Biol Chem. 1992 Apr 15;267(11):7381–7385. [PubMed] [Google Scholar]
  9. Hein D. W., Ferguson R. J., Doll M. A., Rustan T. D., Gray K. Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant wild-type, mutant, and chimeric NAT2 allozymes. Hum Mol Genet. 1994 May;3(5):729–734. doi: 10.1093/hmg/3.5.729. [DOI] [PubMed] [Google Scholar]
  10. Hickman D., Palamanda J. R., Unadkat J. D., Sim E. Enzyme kinetic properties of human recombinant arylamine N-acetyltransferase 2 allotypic variants expressed in Escherichia coli. Biochem Pharmacol. 1995 Aug 25;50(5):697–703. doi: 10.1016/0006-2952(95)00182-y. [DOI] [PubMed] [Google Scholar]
  11. KITZ R., WILSON I. B. Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J Biol Chem. 1962 Oct;237:3245–3249. [PubMed] [Google Scholar]
  12. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  13. Ma Y. Z., Tsou C. L. Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride. Biochem J. 1991 Jul 1;277(Pt 1):207–211. doi: 10.1042/bj2770207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Riddle B., Jencks W. P. Acetyl-coenzyme A: arylamine N-acetyltransferase. Role of the acetyl-enzyme intermediate and the effects of substituents on the rate. J Biol Chem. 1971 May 25;246(10):3250–3258. [PubMed] [Google Scholar]
  15. Takahashi K. The reaction of phenylglyoxal with arginine residues in proteins. J Biol Chem. 1968 Dec 10;243(23):6171–6179. [PubMed] [Google Scholar]
  16. Vatsis K. P., Weber W. W., Bell D. A., Dupret J. M., Evans D. A., Grant D. M., Hein D. W., Lin H. J., Meyer U. A., Relling M. V. Nomenclature for N-acetyltransferases. Pharmacogenetics. 1995 Feb;5(1):1–17. doi: 10.1097/00008571-199502000-00001. [DOI] [PubMed] [Google Scholar]
  17. Watanabe M., Igarashi T., Kaminuma T., Sofuni T., Nohmi T. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes. Environ Health Perspect. 1994 Oct;102 (Suppl 6):83–89. doi: 10.1289/ehp.94102s683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Watanabe M., Sofuni T., Nohmi T. 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. J Biol Chem. 1992 Apr 25;267(12):8429–8436. [PubMed] [Google Scholar]
  19. Waterman M. S. Multiple sequence alignment by consensus. Nucleic Acids Res. 1986 Nov 25;14(22):9095–9102. doi: 10.1093/nar/14.22.9095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weber W. W., Cohen S. N. N-acetylation of drugs: isolation and properties of an N-acetyltransferase from rabbit liver. Mol Pharmacol. 1967 May;3(3):266–273. [PubMed] [Google Scholar]
  21. Wu D., Hersh L. B. Identification of an active site arginine in rat choline acetyltransferase by alanine scanning mutagenesis. J Biol Chem. 1995 Dec 8;270(49):29111–29116. doi: 10.1074/jbc.270.49.29111. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES