Abstract
Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH 3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH 3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.
Highlights
Arylamine N-acetyltransferases (NATs) are well characterised xenobiotic-metabolising enzymes which catalyse the acetyl Coenzyme A (AcCoA)-dependent N, O- and N,O-acetylation of aromatic amines and hydrazines
The third set of conditions demonstrated that (MOUSE)NAT2 could catalyse the time-dependent generation of CoA from AcCoA in the presence of folate but that no acetylation of folate occurred, at least under the single time-point conditions used. These results suggest a possible role of folate as a cofactor for AcCoA hydrolysis by both (HUMAN)NAT1 and (MOUSE)NAT2, rather than an acetylation substrate
The experiments described in this paper demonstrate that (HUMAN)NAT1 and its homologue (MOUSE)NAT2 can catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate
Summary
Arylamine N-acetyltransferases (NATs) are well characterised xenobiotic-metabolising enzymes which catalyse the acetyl Coenzyme A (AcCoA)-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines. They have been shown to play important endogenous roles as well as having potential as novel targets for pharmacological intervention [1,2]. The human genome contains two adjacent NAT genes and a pseudogene (NATP) Both functional genes encode enzymes which catalyse the transfer of an acetyl group from AcCoA to an arylamine resulting in the formation of an amide bond, their individual substrate specificities differ [2,3]. Specific combinations of single nucleotide polymorphisms (SNPs) in the (HUMAN)NAT2 gene are associated with slow acetylation of a number of therapeutic agents as well as industrial chemicals such as arylamines
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