title: The role of lysine(100) in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: implications for other acetyltransferases.
abstract: The arylamine N-acetyltransferases (NATs) catalyze the acetylation of aromatic and heterocyclic amines as well as hydrazines. All proteins in this family of enzymes utilize acetyl coenzyme A (AcCoA) as an acetyl donor, which initially binds to the enzyme and transfers an acetyl group to an active site cysteine. Here, we have investigated the role of a highly conserved amino acid (Lys(100)) in the enzymatic activity of human NAT1. Mutation of Lys(100) to either a glutamine or a leucine significantly increased the Ka for AcCoA without changing the Kb for the acetyl acceptor p-aminobenzoic acid. In addition, substrate inhibition was more marked with the mutant enzymes. Steady state kinetic analyzes suggested that mutation of Lys(100) to either leucine or glutamine resulted in a less stable enzyme-cofactor complex, which was not seen with a positively charged arginine at this position. When p-nitrophenylacetate was used as acetyl donor, no differences were seen between the wild-type and mutant enzymes because p-nitrophenylacetate is too small to interact with Lys(100) when bound to the active site. Using 3'-dephospho-AcCoA as the acetyl donor, kinetic data confirmed that Ly(100) interacts with the 3'-phosphoanion to stabilize the enzyme-cofactor complex. Mutation of Lys(100) decreases the affinity of AcCoA for the protein and increases the rate of CoA release. Crystal structures of several other unrelated acetyltransferases show a lysine or arginine residue within 3Å of the 3'-phosphoanion of AcCoA, suggesting that this mechanism for stabilizing the complex by the formation of a salt bridge may be widely applicable in nature.
authors: Laboratory for Molecular and Cellular Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: r.minchin@uq.edu.au.
authors: Rodney F
authors: RF
authors: Minchin
authors: Laboratory for Molecular and Cellular Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors: Neville J
authors: NJ
authors: Butcher
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000107:Acetylation; D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D007527:Isoenzymes; D000105:Acetyl Coenzyme A; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D008239:Lysine
keywords: Acetylcoenzyme A; Arylamine N-acetyltransferase; Kinetics; Lysine; Mutagenesis