Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa.
BACKGROUND
Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.


RESULTS
We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations.


CONCLUSIONS
This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.
Westwood
Isaac M
IM
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK. isaac.westwood@pharm.ox.ac.uk <isaac.westwood@pharm.ox.ac.uk>
Sim
Edith
E
D000105:Acetyl Coenzyme A / Q000378:metabolism; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D019804:Mesalamine / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D004791:Enzyme Inhibitors; D019804:Mesalamine; D000105:Acetyl Coenzyme A; D001191:Arylamine N-Acetyltransferase