The role of lysine100 in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: Implications for other acetyltransferases

Minchin, Rodney F and Butcher, Neville J (2015) The role of lysine100 in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: Implications for other acetyltransferases. Biochemical Pharmacology, 94 3: 195-202. doi:10.1016/j.bcp.2015.01.015

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Author Minchin, Rodney F
Butcher, Neville J
Title The role of lysine100 in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: Implications for other acetyltransferases
Journal name Biochemical Pharmacology   Check publisher's open access policy
ISSN 1873-2968
0006-2952
Publication date 2015-04-01
Year available 2015
Sub-type Article (original research)
DOI 10.1016/j.bcp.2015.01.015
Open Access Status File (Author Post-print)
Volume 94
Issue 3
Start page 195
End page 202
Total pages 8
Place of publication Philadelphia, United States
Publisher Elsevier Inc
Collection year 2016
Language eng
Formatted 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 3 Å 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.
Keyword Arylamine N-acetyltransferase
Kinetics
Mutagenesis
Acetylcoenzyme A
Lysine
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
School of Biomedical Sciences Publications
 
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