PURIFICATION OF RECOMBINANT HUMAN N-ACETYLTRANSFERASE TYPE-1 (NAT1) EXPRESSED IN ESCHERICHIA-COLI AND CHARACTERIZATION OF ITS POTENTIAL ROLE IN FOLATE METABOLISM

Human arylamine N-acetyltransferase type 1 (NAT1) has been cloned from human genomic DNA, into the vector pET(5a) and expressed in Escherich ia coli. The recombinant protein has been purified to apparent homogen eity using anion exchange chromatography. The arylamine acceptor speci ficity, and the effect of potential NAT1 inhibitors has been investiga ted using purified recombinant protein. The K-m of the recombinant NAT 1 protein for the substrates para-aminobenzoate (p-aba) and 4-aminosal icylate are 14.3 and 11.8 mu M, respectively. Folate and amethopterin were found to be potent competitive inhibitors of p-aba acetylation, w ith K-i values of 13.3 and 9.5 mu M, respectively. The pteroate moiety of folate, in contrast is a poor inhibitor, with 100 mu M pteroate in hibiting only 40% of NAT1 activity. A catabolite of folate para-aminob enzoly-L-glutamate has also been shown to be a NAT1 substrate with a K -m value of 263 mu M.