MECHANISTIC INSIGHTS INTO THE CATALYTIC MECHANISM AND INHIBITION OF MYCOBACTERIUM TUBERCULOSIS ISOCITRATE LYASE
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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. ICL which is involved in the first committed step of the glyoxylate cycle have been extensively characterized, both biochemically and structurally. The importance of such knowledge for the development of new inhibitors against M. tuberculosis ICL has been the inspiration for this thesis.ICL catalyzes the reversible retro-aldol cleavage of isocitrate into succinate and glyoxylate. Previous studies provided evidence for the involvement of two proton transfer steps. Current study aims to reveal the essential residues responsible for the acid–base catalysis by MtICL via mutagenic and kinetic isotope effect (KIE) experiments. Cys191 and His193 were mutated to probe the impact of these residues on formation of the purported enolate intermediate, while Lys189, His180 and Tyr89 were mutated to evaluate their possible role as acid residues for proton transfer to the oxygen that becomes the alcohol in isocitrate. Cys191 and His193 was found to be critical for the catalytic activity as its mutation reduced the kcat by almost 50,000 times than that of the wild type enzyme. Moreover, mutation of these residues resulted in an intrinsic succinate KIEs that is attributed to its role in stabilizing the enolate-like transition state. 3-Bromopyruvate, an inhibitor that forms a covalent bond with Cys191 displayed an inverse solvent isotope effect of 0.7 ± 0.4 on kinact/KI . These results support the hypothesis of an unfavorable prebinding isomerization of the active site Cys191−His193 pair to the thiolate− imidazolium form, a process that is favored in D2O. Mutation of His180 and Try89 showed relatively less hindrance to catalysis as compared to Lys189 which reduced the catalytic activity >2500 fold as compared to the wild type enzyme. A noticeable increase in primary KIEs measured for deuterated succinate from 2.1 (kcat) and 2.7 (kcat/Km) to 4.5 and 3.3 respectively, upon mutation of Lys189, prompted us to propose a new concerted retro-aldol mechanism for MtICL. This investigation broadens our understanding of the role of active site residues in enzymatic mechanism. Insights gained on the enzyme’s reaction mechanism can aid in design of transition state analogues for the inhibition of MtICL3-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 100 times faster than 3-NP at pH 7.5. Through jump-dilution kinetics, we revealed that P3N is an irreversible inhibitor. 13C-NMR, mass spectrometric analysis and X-ray crystallographic characterization confirms the formation of an unprecedented thiohydroximate adduct preferably through a nitronate intermediate. An inverse solvent isotope effect (SIE) of 0.55 ± 0.05 , relatively large KIE of 3.5 ± 0.5 on deuterated 3-NP and a buffer dependency seen on kinact/KI for 3-NP led us to believe that the deprotonation of 3-NP is the slow rate limiting step catalyzed by the buffer in the process of formation of the P3N intermediate. The transition from an inverse SIE for 3-NP to normal SIE for P3N indicates the presence of another solvent dependent step during the formation of the covalent adduct. These mechanistic knowledge of inactivation of MtICL with 3-NP may spur new innovative designs to develop covalent inhibitors for this enzyme.