title: Purification of recombinant human N-acetyltransferase type 1 (NAT1) expressed in E. coli and characterization of its potential role in folate metabolism.
abstract: Human arylamine N-acetyltransferase type 1 (NAT1) has been cloned from human genomic DNA, into the vector pET(5a) and expressed in Escherichia coli. The recombinant protein has been purified to apparent homogeneity using anion exchange chromatography. The arylamine acceptor specificity, and the effect of potential NAT1 inhibitors has been investigated using purified recombinant protein. The Km of the recombinant NAT1 protein for the substrates para-aminobenzoate (p-aba) and 4-aminosalicylate are 14.3 and 11.8 microM, respectively. Folate and amethopterin were found to be potent competitive inhibitors of p-aba acetylation, with Ki values of 13.3 and 9.5 microM, respectively. The pteroate moiety of folate, in contrast is a poor inhibitor, with 100 microM pteroate inhibiting only 40% of NAT1 activity. A catabolite of folate para-aminobenzoly-L-glutamate has also been shown to be a NAT1 substrate with a Km value of 263 microM.
authors.affiliation: Department of Pharmacology, University of Oxford, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Ward
authors.forename: M J
authors.initials: MJ
authors.lastname: Summers
authors.forename: E
authors.initials: E
authors.lastname: Sim
mesh_terms: D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D005492:Folic Acid; D001191:Arylamine N-Acetyltransferase