NMR investigation of the catalytic mechanism of arylamine N-acetyltransferase from Salmonella typhimurium.
Arylamine N-acetyltransferases (NAT) are a family of enzymes found in both eucaryotes and procaryotes, which catalyse the N-acetylation of a range of arylamine and hydrazine drugs and carcinogenic arylamines, using acetyl Coenzyme A as a cofactor. Here we describe a nuclear magnetic resonance (NMR) investigation of the interaction of substrates with Salmonella typhimurium NAT. For solution NMR investigations, pure recombinant NAT from S. typhimurium was used at up to 0.1 mM. We demonstrate that a hydrazine substrate, isoniazid (INH), binds to the protein in the absence of the cofactor, acetyl CoA, and thereby suggest that even though the catalysis may follow a ping-pong pathway, ligand-enzyme interactions can occur in the absence of acetyl CoA.
Delgoda
Rupika
R
Department of Pharmacology, University of Oxford, Mansfield Road, UK.
Lian
Lu Yun
LY
Sandy
James
J
Sim
Edith
E
D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D007538:Isoniazid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D001191:Arylamine N-Acetyltransferase; D003065:Coenzyme A; D007538:Isoniazid