Isocitrate lyase from Mycobacterium tuberculosis: Mechanistic insights and kinetic isotope effects
Moynihan, Margaret M.
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Isocitrate lyase from Mycobacterium tuberculosis has been studied in order to design inhibitors for new antitubercular therapies. Steady-state kinetics was determined in both the forward and reverse direction and its kinetic mechanism established. The chemical mechanism, which reversibly cleaves isocitrate into succinate and glyoxylate, involves two proton-transfer steps and the enzyme residues responsible have been determined. Through solvent kinetic isotope effect (KIE) studies, Cys191 was identified as the general base that deprotonates succinate in the isocitrate synthesis direction. A large inverse solvent KIE of 0.56 was observed on k cat / K m , stemming from an equilibrium isotope effect. This is attributed to an unfavorable pre-binding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is less unfavorable in D2O. This was also supported by the observed solvent KIE of 0.40 on the association of the succinate analog 3-nitropropionate, demonstrating that it is a reversible mechanism-based inhibitor that is converted to its more-potent nitronate form by Cys191. Mutation of Cys191 led to completely inactive enzyme while mutation of His193 displayed a 50,000-fold decrease in k cat . Additionally, primary KIEs measured for deuterated succinate increased from 2.1 ( k cat ) and 2.7 ( k cat / K m ) to 6.0 and 6.9, respectively, upon mutation of His193, indicating that it also plays a critical role in deprotonation of succinate. The residue involved in the second proton-transfer step was also probed using mutagenesis and succinate deuterium KIEs. Two residues, Tyr89 and His180, proximal to the carbonyl oxygen of glyoxylate were mutated. Both mutations negatively impacted steady-state kinetic parameters but neither severely enough to identify conclusively the general acid involved in protonation of the hypothetical alkoxide intermediate. Because the second proton-transfer step is hypothesized to be reflected in k cat and not k cat / K m , mutation of the general acid residue should decrease the succinate deuterium KIE on k cat while leaving the KIE on k cat / K m unchanged. This result was observed with H180A but not with Y89F, which instead yielded a larger KIE on k cat / K m and an unchanged KIE on k cat . Therefore, His180 is implicated as the general acid in the synthesis of isocitrate. Finally, succinate and glyoxylate were synthesized with 13 C labeled at various positions for 13 C KIE determination. A suitable coupling system was developed and preliminary 13 C-NMR experiments performed in an attempt to determine the KIEs for carbon-carbon bond formation. Altogether, the 13 C and 2H KIEs arising from this step and the two proton-transfer steps lay the foundation for transition state analysis, which is required for the design of transition state analogs as possible potent inhibitors of Mt ICL.