Characterization of Naturally Occurring and Recombinant HumanN-Acetyltransferase Variants Encoded byNAT1

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.