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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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Related in: MedlinePlus

A comparison of the C-terminal residues of STNAT and PANAT. The active-site triad residues of PANAT (Cys-His-Asp) and the C-terminal residues of PANAT (purple) and STNAT (black) are shown in ball and stick representation. The distances between the active site Cys sulfur atom and residues Leu276 and Phe273 from PANAT and STNAT respectively are 25.8 Å and 17.6 Å respectively, and were determined by using SwissPDB Viewer [46]. The figure was produced with Aesop (M. E. M. Noble, unpublished results).
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Figure 5: A comparison of the C-terminal residues of STNAT and PANAT. The active-site triad residues of PANAT (Cys-His-Asp) and the C-terminal residues of PANAT (purple) and STNAT (black) are shown in ball and stick representation. The distances between the active site Cys sulfur atom and residues Leu276 and Phe273 from PANAT and STNAT respectively are 25.8 Å and 17.6 Å respectively, and were determined by using SwissPDB Viewer [46]. The figure was produced with Aesop (M. E. M. Noble, unpublished results).

Mentions: The Km value for AcCoA with PANAT is significantly larger than the Km,app value for AcCoA with the STNAT protein [38]. However, the two C-terminal truncation mutants of STNAT, reported by Mushtaq and colleagues [38], have apparent Michaelis constants which are very similar to the Km for AcCoA with PANAT. The STNAT sequence has eight more residues at the C-terminus than the PANAT sequence, and this C-terminal section of the STNAT is responsible for the observed differences in the truncation mutants compared with the full-length protein [38]. The comparatively shorter length of the C-terminus of PANAT may therefore be responsible for the higher apparent Km for AcCoA. The X-ray crystal structures of the two proteins are available, and a comparison of the C-terminal residues of these proteins indicates that a cleft which leads to the active site is blocked by the C-terminus of STNAT relative to that of PANAT (Figure 5). The terminal residue (Leu276) in the PANAT structure is 25.8 Å from the active site cysteine, compared to 17.6 Å between Phe273 and the active site Cys in STNAT.


Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa.

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

A comparison of the C-terminal residues of STNAT and PANAT. The active-site triad residues of PANAT (Cys-His-Asp) and the C-terminal residues of PANAT (purple) and STNAT (black) are shown in ball and stick representation. The distances between the active site Cys sulfur atom and residues Leu276 and Phe273 from PANAT and STNAT respectively are 25.8 Å and 17.6 Å respectively, and were determined by using SwissPDB Viewer [46]. The figure was produced with Aesop (M. E. M. Noble, unpublished results).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: A comparison of the C-terminal residues of STNAT and PANAT. The active-site triad residues of PANAT (Cys-His-Asp) and the C-terminal residues of PANAT (purple) and STNAT (black) are shown in ball and stick representation. The distances between the active site Cys sulfur atom and residues Leu276 and Phe273 from PANAT and STNAT respectively are 25.8 Å and 17.6 Å respectively, and were determined by using SwissPDB Viewer [46]. The figure was produced with Aesop (M. E. M. Noble, unpublished results).
Mentions: The Km value for AcCoA with PANAT is significantly larger than the Km,app value for AcCoA with the STNAT protein [38]. However, the two C-terminal truncation mutants of STNAT, reported by Mushtaq and colleagues [38], have apparent Michaelis constants which are very similar to the Km for AcCoA with PANAT. The STNAT sequence has eight more residues at the C-terminus than the PANAT sequence, and this C-terminal section of the STNAT is responsible for the observed differences in the truncation mutants compared with the full-length protein [38]. The comparatively shorter length of the C-terminus of PANAT may therefore be responsible for the higher apparent Km for AcCoA. The X-ray crystal structures of the two proteins are available, and a comparison of the C-terminal residues of these proteins indicates that a cleft which leads to the active site is blocked by the C-terminus of STNAT relative to that of PANAT (Figure 5). The terminal residue (Leu276) in the PANAT structure is 25.8 Å from the active site cysteine, compared to 17.6 Å between Phe273 and the active site Cys in STNAT.

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