STRUCTURE-FUNCTION STUDIES OF HUMAN ARYLAMINE N-ACETYLTRANSFERASES NAT1 AND NAT2 - FUNCTIONAL-ANALYSIS OF RECOMBINANT NAT1 NAT2 CHIMERAS EXPRESSED IN ESCHERICHIA-COLI/

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the bi otransformation of primary aromatic amine or hydrazine drugs and xenob iotics. These enzymes share 81% amino acid sequence identity, yet diff er markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large r egions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease diges tions and fragment religation into the tao promoter-based phagemid pKE N2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for hetero logous expression in Escherichia coil. Induction of hybrid gene expres sion in recombinant transformants of E. coil strain XA90 led to the pr oduction of soluble, catalytically active acetylating enzymes in all c ases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constant s (apparent K-m, V-max, and V-max/K-m) for the NAT1-selective and NAT2 -selective substrates p-aminosalicylic acid and sulfamethazine, respec tively, and for their in vitro stabilities at 37 degrees C. The ratio of the V-max/K-m for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NA T1 or NAT2 type, except for one novel chimera possessing a low Michael is constant and a high maximal velocity for the acetylation of both su bstrates. A central region (amino acids 112-210) within the 290-residu e polypeptide appeared to play a role in determining NAT1- or NAT2-typ e behavior. On the other hand, the region (residues 47-111) encompassi ng the putative active site cysteine (Cys(68)) was important in contri buting to a low apparent K-m for p-aminosalicylic acid but not for sul famethazine, while amino acids 211-250 affected K-m for sulfamethazine and 251-290 influenced K-m for both substrates. Maximal velocities we re highest for both substrates when the central 112-210 amino acid reg ion was derived from NAT1. Finally, the region from amino acids 211-25 0 in NAT2 was important in determining its greater intrinsic enzyme st ability than that exhibited by NAT1.