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

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 (NAT14) and the three mutant alleles (NAT110, 11, and 16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT14/4 and NAT110/10 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 NAT110 affected none of the aforementioned parameters evaluated both with recombinant NAT110 and with the naturally occurring allele. Function was also unaffected by the coding and 3' noncoding exon mutations in NAT111. In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3' untranslated region of NAT116 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.