Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa

Isaac M Westwood,Edith Sim

doi:10.1186/1471-2091-8-3

Abstract

BackgroundArylamine 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.ResultsWe 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.ConclusionThis 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.

Highlights

Arylamine N-acetyltransferases (NATs) are important drug- and carcinogenmetabolising 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
For the series b = 1, reaction velocities (Va,b) are measured in the presence of a constant concentration of 5-aminosalicylic acid (B), and a series of actual concentrations of acetyl coenzyme A (AcCoA) which are described as multiples of the arbitrarily fixed concentration A
For the series a = 1, Va,b values are measured in the presence of AcCoA and a series of concentrations of 5-aminosalicylic acid

Summary

Introduction

Arylamine N-acetyltransferases (NATs) are important drug- and carcinogenmetabolising 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. NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is of interest as a potential drug target. NATs catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A (AcCoA) to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. The precise endogenous function of NAT in mycobacteria has not been established, genetic studies suggest strongly that NAT has a role in cell wall complex lipid biosynthesis in Mycobacterium bovis BCG [3]. It has been proposed that NAT represents a good anti-tubercular (page number not for citation purposes)

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