Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis

Alison L. Sikora, Brenda A. Frankel, John S. Blanchard

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the front-line antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of M. tuberculosis NAT which reveals that substituted anilines are excellent substrates but that isoniazid is a very poor substrate for this enzyme. We propose that the expression of NAT from M. tuberculosis (TBNAT) is unlikely to be a significant cause of isoniazid resistance. The kinetic parameters for a variety of TBNAT substrates were examined, including 3-amino-4-hydroxybenzoic acid and AcCoA, revealing K m values of 0.32 ± 0.03 and 0.14 ± 0.02 mM, respectively. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a bi-bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters reveals that one enzyme group must be deprotonated for optimal catalytic activity and that two amino acid residues at the active site of the free enzyme are involved in binding and/or catalysis. Solvent kinetic isotope effects suggest that proton transfer steps are not rate-limiting in the overall reaction for substituted aniline substrates but become rate-limiting when poor hydrazide substrates are used.

Original languageEnglish (US)
Pages (from-to)10781-10789
Number of pages9
JournalBiochemistry
Volume47
Issue number40
DOIs
StatePublished - Oct 7 2008

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Arylamine N-Acetyltransferase
Mycobacterium tuberculosis
Acetyltransferases
Isoniazid
Kinetics
Substrates
Enzymes
Acetyl Coenzyme A
Kinetic parameters
Aniline Compounds
Hydrazines
Antitubercular Agents
Proton transfer
Mycobacterium smegmatis
Prodrugs
Isotopes
Catalysis
Catalyst activity
Protons
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis. / Sikora, Alison L.; Frankel, Brenda A.; Blanchard, John S.

In: Biochemistry, Vol. 47, No. 40, 07.10.2008, p. 10781-10789.

Research output: Contribution to journalArticle

Sikora, Alison L. ; Frankel, Brenda A. ; Blanchard, John S. / Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis. In: Biochemistry. 2008 ; Vol. 47, No. 40. pp. 10781-10789.
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