ABSTRACT
Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.
Subject(s)
Bacterial Proteins/genetics , Isocitrate Lyase/genetics , Isocitrates/chemistry , Mycobacterium tuberculosis/enzymology , Tuberculosis/drug therapy , Bacterial Proteins/antagonists & inhibitors , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Glyoxylates/chemistry , Humans , Isocitrate Lyase/antagonists & inhibitors , Ligands , Malates/chemistry , Microscopy, Fluorescence , Molecular Docking Simulation , Spectrometry, Mass, Electrospray Ionization , Succinic Acid/chemistry , Sulfhydryl Compounds/chemistry , Tuberculosis/microbiology , Tuberculosis/prevention & controlABSTRACT
Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for (D2O)V and 0.56 ± 0.07 for (D2O)(V/Ksuccinate). Whereas the (D2O)V is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse (D2O)(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is saturated with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analogue, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 µM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for association and unity for dissociation. In contrast, itaconate, a succinate analogue lacking an acidic α-proton, did not display slow-binding behavior and yielded a (D2O)Ki of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymatically.
Subject(s)
Cysteine/chemistry , Isocitrate Lyase/antagonists & inhibitors , Isocitrate Lyase/metabolism , Nitro Compounds/pharmacology , Propionates/pharmacology , Catalysis , Deuterium Oxide , Hydrogen-Ion Concentration , Kinetics , Mycobacterium tuberculosis/enzymology , Solvents , Succinates/chemistry , Succinates/pharmacologyABSTRACT
OBJECTIVE: To determine if low-flow nasal prongs therapy with room air, compared with no treatment, facilitates weaning from nasal continuous positive airway pressure (NCPAP) in very low birth weight (VLBW, birth weight <1500 g) infants. STUDY DESIGN: VLBW infants who received respiratory support for ≥ 48 hours and who were stable on NCPAP for 24 hours were eligible for inclusion in this multicenter, randomized controlled trial. On stopping NCPAP, infants were randomized to receive 1 L/min air via nasal prongs or to spontaneous breathing in room air. The primary outcome measure was failure to wean. Secondary outcome measures included length of time to failure and change in heart rate, respiratory rate, oxygen saturation, and respiratory distress score. RESULTS: Seventy-eight infants were randomized: 39 to nasal prongs and 39 to spontaneous breathing. The groups were similar at birth and at randomization. Sixteen infants (41%) in the nasal prongs group failed the weaning process compared with 12 infants (31%) in the spontaneous breathing group (OR 1.57, 95% CI 0.56 to 4.43, P = .48). There were no significant differences between the groups in secondary outcomes. CONCLUSIONS: In this study, we did not demonstrate a benefit of low-flow room air via nasal prongs to wean VLBW infants from NCPAP.
