Biochemical evidence that InhA is the target for isoniazid

Enzymatic characterization of recombinant purified InhA enzyme showed that InhA is involved in fatty acid elongation and/or mycolic acid synthesis. It is an enoyl-ACP reductase that catalyses the NADH-dependent reduction of the double bond at position two of the fatty acid which is linked to the acyl carrier protein (ACP) part of the type II fatty acid synthesis system (Quemard et al 1995). Kinetic analysis of InhA indicated that it preferentially reduces long fatty acyl substrates (at least 16 carbon atoms) consistent with the proposed role for InhA in synthesizing very long-chain fatty acids in mycolic acid synthesis.

Isoniazid inhibits the InhA enzyme by a mechanism that requires NADH, Mn2+ and oxygen (Basso et al 1996, Johnsson et al 1995, Zabinski & Blanchard 1997). Inhibition was demonstrated using an in vitro reaction system with purified InhA enzyme; the combination of isoniazid, NADH and Mn2+ caused the loss of InhA activity (Fig. 2). KatG accelerates the inhibition process perhaps by oxidizing Mn2+ to form Mn3+ (Magliozzo & Marcinkeviciene 1997). Structural studies in which InhA crystals were formed in the presence of isoniazid, NADH and Mn2+(3+) clearly showed that isoniazid was covalently attached to the nicotinamide ring of NADH at the site of the hydride exchange (isonicotinic acyl-NADH; Fig. 3; Rozwarski et al 1998). The isonicotinic acyl-NADH was bound to InhA in the NADH-binding site, forming a close association with amino acids and a structural arrangement that is apt to have a higher affinity than NADH bound to InhA. Rozwarski et al (1988) proposed that the isonicotinic acyl-NADH forms by the addition of an isonicotinic acyl radical to an NAD. radical or

FIG. 2. Inhibition of InhA by isoniazid. InhA (3 ^M) was incubated in 100 mM phosphate buffer, pH 7.5 at 25°. Solid lines: mixtures included NADH (100 ^M), isoniazid (100 ^M) and MnCl2 (1 ^M) with KatG (1 ^M; filled circles) or without KatG (open circles). The dashed line represents results from control experiments in which mixtures lacking MnCl2, NADH or isoniazid showed no inhibition of InhA (Zabinski & Blanchard 1997, Basso et al 1996). Adapted from Zabinski & Blanchard (1997) and reprinted with permission (Copyright 1997 American Chemical Society).

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FIG. 2. Inhibition of InhA by isoniazid. InhA (3 ^M) was incubated in 100 mM phosphate buffer, pH 7.5 at 25°. Solid lines: mixtures included NADH (100 ^M), isoniazid (100 ^M) and MnCl2 (1 ^M) with KatG (1 ^M; filled circles) or without KatG (open circles). The dashed line represents results from control experiments in which mixtures lacking MnCl2, NADH or isoniazid showed no inhibition of InhA (Zabinski & Blanchard 1997, Basso et al 1996). Adapted from Zabinski & Blanchard (1997) and reprinted with permission (Copyright 1997 American Chemical Society).

by an isonicotinic acyl anion to an NAD+ (Fig. 3). Isonicotinic acyl-NADH forms within the InhA active site.

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