Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl- CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenob iotics and their Nhydroxylated metabolites. We previously used a panel of r ecombinant NAT1 /NAT2 chimaeric proteins to identify linear amino acid segm ents that have roles in imparting the distinct catalytic specificities to t hese proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine r esidue Cys(68) was important in determining NAT substrate selectivity. In t he present study we have refined our analysis through further chimaera gene ration of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme kinetic analysis of these mutant protein s with the NAT1-selective and NAT2-selective substrates p-aminosalicylic ac id (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrat e selectivity. Modification of Arg(127) had the greatest effect on specific ity for PAS, whereas changing Phe(125) had the greatest effect on specifici ty for SMZ. Selected NAT mutants exhibited K-m values for acetyl-CoA that w ere comparable with those of the wild-type NATs, implying that the mutation s affected acceptor substrate specificity rather than cofactor binding affi nity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active- site pocket surrounding Cys(68) and function as important determinants of N AT substrate selectivity.