Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis.
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.
Sikora
Alison L
AL
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Frankel
Brenda A
BA
Blanchard
John S
JS
D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001191:Arylamine N-Acetyltransferase
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