The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics. These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli. Induction of hybrid gene expression in recombinant transformants of E. coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C. The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates. A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior. On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates. Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1. Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.