MOLECULAR-GENETICS OF HUMAN POLYMORPHIC N-ACETYLTRANSFERASE - ENZYMATIC ANALYSIS OF 15 RECOMBINANT WILD-TYPE, MUTANT, AND CHIMERIC NAT2 ALLOZYMES

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylati on of arylamine drugs and carcinogens. Human acetylator phenotype is r egulated at the NAT2 locus and has been associated with differential r isk to certain drug toxicities or cancer. We examined arylamine substr ate and acetyl coenzyme A cofactor affinities, and the N-acetyltransfe rase catalytic activities of the wild-type and 14 different mutant or chimeric human NAT2 alleles expressed in an Escherichia coli JM 105 ex pression system. NAT2 alleles contained nucleic acid substitutions at positions 191(G --> A; Arg(64) --> Gln), 282(C --> T; silent), 341(T - -> C; Ile(114) --> Thr), 481(C --> T; silent), 590(G --> A; Arg(197) - -> Gln), 803(A --> G; Lys(268) --> Arg), 857(G --> A; Gly(286) --> Glu ) and various combinations (282/590; 282/803; 282/857; 341/481; 341/80 3; 341/481/803; 481/803) of the 870 base pair NAT2 coding region. Expr ession of all 15 NAT2 alleles produced immunoreactive NAT2 protein wit h N-acetylation activity. NAT2 proteins encoded by alleles with nuclei c acid substitutions at positions 191, 341, 590, 282/590, 341/481, 341 /803, and 341/481/803 exhibited arylamine N-acetyltransferase maximum velocities significantly (P < 0.001) lower than the wild-type NAT2. Th us, nucleic acid substitutions at positions 191, 341, and 590 either a lone or in combination with other silent or conservative amino acid su bstitutions were sufficient to result in NAT2 proteins with significan t reductions in N-acetylation activities. The recombinant NAT2 protein s also showed relative differences in intrinsic stability following in cubation at 37 degrees C and 50 degrees C. NAT2 encoded by alleles wit h nucleotide substitutions at positions 191 and 857 were particularly unstable relative to the wild type. These findings using recombinant N AT2 allozymes provide important insight into the molecular genetic bas is for human slow acetylator phenotype.