Allosteric regulation of arylamine N-acetyltransferase 1 by adenosine triphosphate

Abstract

In the present study, a screen of adenosine analogs as potential modulators of arylamine-Nacetyltransferase 1 activity identified ATP as an inhibitor within its range of physiological concentrations. Kinetically, ATP was a non-competitive inhibitor with respect to the acetyl acceptor but a competitive inhibitor with respect to the acetyl donor (acetyl-coenzyme A). In silico modelling predicted that ATP bound within the active site cleft arranged with the triphosphate group in close proximity to arginine 127. Since lysine 100 has previously been implicated in the binding of acetyl-coenzyme A to the enzyme, this amino acid was mutated to either an arginine or a glutamine. Both substitutions significantly changed the affinity of ATP for the enzyme, as well as the nature of the interaction to one with a large Hill coefficient (>3). Under these conditions, ATP was a strong allosteric modulator of arylamine-Nacetyltransferase 1 activity. Western blot analysis identified lysine 100 as a site of posttranslational modification by acetylation. The results suggest that acetylation of lysine 100 converts arylamine-N-acetyltransferase 1 into a switch modulated by ATP. This observation provides important understanding of the molecular regulation of NAT1 activity and may reveal possible insight into the endogenous role of the enzyme.

Modulation of Human Arylamine N-Acetyltransferase 1 Activity by Lysine Acetylation: Role of p300/CREB-Binding Protein and Sirtuins 1 and 2

“…The positions of p300 and NAT1 are shown. p300/CBP and SIRT1/2 Regulate NAT1 Acetylation (Minchin et al, 2018). In cells transfected with NAT1 alone, ATP (1 mM) decreased enzymatic activity in cytosolic preparations by 40% from 35 6 3 to 21 6 0.5 nmol/min per density unit (Fig.…”

“…Recent studies have suggested that NAT1 protein modifications regulate enzyme function. For example, alterations to lysine 100 (K 100 ) by site-directed mutagenesis decreased the affinity of the acetyl donor acetyl coenzyme A (AcCoA) for the protein (Minchin et al, 2018), suggesting it was important for AcCoA binding in the active site (Minchin and Butcher, 2015). K 100 has also been associated with ATP binding to NAT1 (Minchin et al, 2018).…”

“…For example, alterations to lysine 100 (K 100 ) by site-directed mutagenesis decreased the affinity of the acetyl donor acetyl coenzyme A (AcCoA) for the protein (Minchin et al, 2018), suggesting it was important for AcCoA binding in the active site (Minchin and Butcher, 2015). K 100 has also been associated with ATP binding to NAT1 (Minchin et al, 2018). ATP is an allosteric modulator and a competitive inhibitor of AcCoA binding to the enzyme.…”

“…ATP is an allosteric modulator and a competitive inhibitor of AcCoA binding to the enzyme. Ectopic expression of NAT1 where K 100 was substituted by either arginine or glutamine clearly demonstrated that this amino acid is a target for acetylation (Minchin et al, 2018). Moreover, activators and inhibitors of the sirtuins (SIRTs), a family of protein deacetylases, have been used with peripheral blood mononuclear cells to identify possible effects of protein acetylation on the activity of NAT1 and the closely related NAT2 (Turiján-Espinoza et al, 2018).…”

Population variability of rhesus macaque (Macaca mulatta) NAT1 gene for arylamine N-acetyltransferase 1: Functional effects and comparison with human

“…Both orthologues are controlled by a conserved Sp1-type housekeeping promoter, adjacent to an upstream non-coding exon 11–13 , and are potentially subject to epigenetic regulation 14,15 . Human NAT1 is also controlled by a second, more tissue-specific promoter 16 , and regulation has been shown to implicate steroid hormones 17–20 , micro-RNAs 21,22 , protein polyubiquitinylation 23,24 , as well as acetylation of the enzyme and allosteric modulation by adenosine triphosphate 25,26 . Human NAT1 and rodent NAT2 proteins have been demonstrated to acetylate the same arylamine substrates 27 , including p-aminobenzoylglutamate (pABGlu), a catabolic derivative of folate 4,27–30 .…”