Over-expression, Purification, and Characterization of Recombinant Human Arylamine N-Acetyltransferase 1

Abstract

Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo.

From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2

“…Recombinant NAT enzymes were prepared as described previously [5], [20], [46]–[53]. Human ((HUMAN)NAT1), murine ((MOUSE)NAT1 and (MOUSE)NAT2) and prokaryotic NATS (from Salmonella typhimurium , Mycobacterium marinum , Mycobacterium smegmatis , Pseudomonas aeruginosa and Mycobacterium tuberculosis ((SALTY)NAT, (MYCMR)NAT, (MYCSM)NAT, (PSEAE)NAT) and (MYCTU)NAT)) were evaluated.…”

Arylamine N‐acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

“…A library of some 5000 compounds was available (Russell et al ., ) and we assembled the other tools required – sufficient of all of the enzymes we wished to use. We produced hundreds of milligrams of each of human NAT1 (Wang et al ., ), hamster NAT2 (Kawamura et al ., ), S. typhimurium NAT (Sinclair et al ., ), M. smegmatis NAT (Sandy et al ., ) and P. aeruginosa NAT (Westwood et al ., ). The mammalian enzymes were to allow any possible toxicity to be identified early but generated very interesting results.…”

Structure and Mechanism of Arylamine N-Acetyltransferases

“…Detailed kinetic and catalytic analyses have been reported to date only for a limited number of enzymes, such as hamster NAT2 [48,67,73], whilst a range of recombinant enzymes has been used for the determination of substrate specific activity profiles, including human NAT1 and NAT2 [43], and the prokaryotic enzymes PANAT [37], STNAT and MSNAT [74]. These studies can assist in furthering understanding of the active site by allowing more detailed structure-activity relationships to be established.…”