Characterization of naturally occurring and recombinant human N-acetyltransferase variants encoded by NAT1

Mol Pharmacol. 2000 Aug;58(2):288-99. doi: 10.1124/mol.58.2.288.

Abstract

The genotype at the NAT1* locus of an interethnic population of 38 unrelated subjects was determined by direct sequencing of 1.6-kb fragments amplified by PCR. The coding exon alone and together with the 3' noncoding exon of the wild-type (NAT1*4) and the three mutant alleles (NAT1*10, *11, and *16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT1*4/*4 and NAT1*10/*10 homozygotes was also isolated. Recombinant and leukocyte cytosolic preparations were thoroughly characterized by N-acetylation activity with several NAT1-specific and -selective substrates, as well as by steady-state kinetics with varying amounts of the substrate (fixed acetyl CoA) and acetyl CoA (fixed substrate), thermodynamics, stability, and protein immunoreactivity with a polyclonal human anti-NAT1. The polyadenylation signal mutation in the 3' noncoding sequence of NAT1*10 affected none of the aforementioned parameters evaluated both with recombinant NAT1*10 and with the naturally occurring allele. Function was also unaffected by the coding and 3' noncoding exon mutations in NAT1*11. In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3' untranslated region of NAT1*16 ostensibly caused disruption of the predicted secondary structure of the pre-mRNA for NAT1 16, culminating in parallel 2-fold decreases in the amount and catalytic activity of NAT1 16 in COS-1 cell cytosol. This novel finding in N-acetylation pharmacogenetics clearly demonstrates a direct link between reduced catalytic activity and structural alteration in the 3' untranslated region of an NAT variant (NAT1*16) brought about by mutation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3' Untranslated Regions / genetics*
  • Acetyltransferases / genetics*
  • Acetyltransferases / metabolism
  • Alleles
  • Animals
  • Arylamine N-Acetyltransferase*
  • COS Cells
  • Escherichia coli
  • Female
  • Gene Expression
  • Genotype
  • Humans
  • Isoenzymes
  • Leukocytes
  • Male
  • Mutation
  • Nucleic Acid Conformation
  • RNA Precursors / chemistry
  • RNA Precursors / metabolism
  • RNA, Messenger / genetics
  • Reading Frames
  • Recombinant Proteins / metabolism

Substances

  • 3' Untranslated Regions
  • Isoenzymes
  • RNA Precursors
  • RNA, Messenger
  • Recombinant Proteins
  • Acetyltransferases
  • Arylamine N-Acetyltransferase
  • N-acetyltransferase 1