Mycobacterium tuberculosis, the causative organism for tuberculosis, is responsible for the highest mortality rate among bacterial pathogens. Persistent M. tuberculosis infections depend on the glyoxylate shunt, a carbon-conserving bypass in the tricarboxylate cycle consisting of isocitrate lyase (ICL) and malate synthase. Since these enzymes are absent in humans but essential to mycobacteria, they are potential drug targets. ICL catalyzes the reversible retro-aldol cleavage of isocitrate into succinate and glyoxylate. 3-Nitropropionate (3-NP) is an analog of succinate that demonstrates slow-onset inhibition of the enzyme, presumably via its conjugate base form, propionate-3-nitronate (P3N). P3N, prepared from 3-NP at pH 13, was found to inhibit the enzyme 70 times faster than 3-NP at pH 7.5. Considering that 0.025% of 3-NP (pKa = 9.1) exists as P3N at this pH, a comparable rate of inhibition was found when the data were replotted against [P3N], strongly supporting P3N as the inhibitory form. Through jump-dilution kinetics, we revealed that P3N is an irreversible inhibitor. Electrospray ionization mass spectrometry showed a time-dependent increase of 100 Da in the mass of ICL following incubation with 3-NP or P3N (119 Da for neutral form), which was accelerated in the presence of glyoxylate. The route of formation for this covalent complex is being exploited using kinetic and solvent isotope effect. The identity of the resulting covalent complex and the mechanism of its formation are the subject of ongoing investigation.

Mechanism of inhibition of Mycobacterium tuberculosis isocitrate lyase by 3-nitropropionate

255th National Meeting (2018)

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