The role of lysine100 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 (Lys100) in the enzymatic activity of human NAT1. Mutation of Lys100 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 Lys100 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 Lys100 when bound to the active site. Using 3′-dephospho-AcCoA as the acetyl donor, kinetic data confirmed that Ly100 interacts with the 3′-phosphoanion to stabilize the enzyme–cofactor complex. Mutation of Lys100 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.