Steady-state kinetics and inhibitory action of antitubercular phenothiazines on mycobacterium tuberculosis type-II NADH-menaquinone oxidoreductase (NDH-2).
Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC50 = 12, 11, and 13 microm, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Takahiro
T
Yano
Lin-Sheng
LS
Li
Edward
E
Weinstein
Jiah-Shin
JS
Teh
Harvey
H
Rubin
D000995:Antitubercular Agents / Q000494:pharmacology*; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010640:Phenothiazines / Q000494:pharmacology*; D011808:Quinone Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011809:Quinones / Q000737:chemistry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D010640:Phenothiazines; D011809:Quinones; D009243:NAD; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D011808:Quinone Reductases; C025204:Ubiquinone Q2