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Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa.

Westwood IM, Sim E - BMC Biochem. (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK. isaac.westwood@pharm.ox.ac.uk <isaac.westwood@pharm.ox.ac.uk>

ABSTRACT

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.

Conclusion: 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.

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The steady-state formation and hydrolysis of the acetyl-enzyme intermediate. The acetyl donor is denoted AcX, where X is p-nitrophenol or CoA. AcNAT refers to the acetylated enzyme intermediate. For a derivation of equation 9, see [34].
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Figure 3: The steady-state formation and hydrolysis of the acetyl-enzyme intermediate. The acetyl donor is denoted AcX, where X is p-nitrophenol or CoA. AcNAT refers to the acetylated enzyme intermediate. For a derivation of equation 9, see [34].

Mentions: In the absence of an acetyl acceptor, the proposed NAT reaction scheme is shown in Figure 3. The proposed scheme is based both on this study and on previously described work with a eukaryotic NAT [34]. Study of the first half of the Ping Pong Bi Bi reaction in this way allows for the determination of the half-life (t1/2) of the acetyl-NAT intermediate, which is a measure of its stability. In the proposed reaction scheme, t1/2 of the acetyl-NAT intermediate is dependent on both k2 and k3 (equations 6 and 7, Figure 3). If the rate of enzyme acetylation is very much faster than the rate of acetyl-enzyme hydrolysis (that is, if k2 is very much larger than k3), then t1/2 of the acetyl-NAT intermediate may be approximated by the following equation: (equations 6–8, Figure 3).


Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa.

Westwood IM, Sim E - BMC Biochem. (2007)

The steady-state formation and hydrolysis of the acetyl-enzyme intermediate. The acetyl donor is denoted AcX, where X is p-nitrophenol or CoA. AcNAT refers to the acetylated enzyme intermediate. For a derivation of equation 9, see [34].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851014&req=5

Figure 3: The steady-state formation and hydrolysis of the acetyl-enzyme intermediate. The acetyl donor is denoted AcX, where X is p-nitrophenol or CoA. AcNAT refers to the acetylated enzyme intermediate. For a derivation of equation 9, see [34].
Mentions: In the absence of an acetyl acceptor, the proposed NAT reaction scheme is shown in Figure 3. The proposed scheme is based both on this study and on previously described work with a eukaryotic NAT [34]. Study of the first half of the Ping Pong Bi Bi reaction in this way allows for the determination of the half-life (t1/2) of the acetyl-NAT intermediate, which is a measure of its stability. In the proposed reaction scheme, t1/2 of the acetyl-NAT intermediate is dependent on both k2 and k3 (equations 6 and 7, Figure 3). If the rate of enzyme acetylation is very much faster than the rate of acetyl-enzyme hydrolysis (that is, if k2 is very much larger than k3), then t1/2 of the acetyl-NAT intermediate may be approximated by the following equation: (equations 6–8, Figure 3).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK. isaac.westwood@pharm.ox.ac.uk <isaac.westwood@pharm.ox.ac.uk>

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus