Vol. 58, Issue 2, 288-299, August 2000

Characterization of Naturally Occurring and Recombinant Human N-Acetyltransferase Variants Encoded by NAT1*

Jesús H. de León,¹ Kostas P. Vatsis,² and Wendell W. Weber

Department of Pharmacology, Medical School, The University of Michigan, Ann Arbor, Michigan

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.