Development of an ESI-LC-MS-Based Assay for Kinetic Evaluation of Mycobacterium tuberculosis Shikimate Kinase Activity and Inhibition

Abstract

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

Slow-Binding Inhibition of Mycobacterium tuberculosis Shikimate Kinase by Manzamine Alkaloids

“…MtSK was expressed and purified following procedures described in a previous report. 31 The purity of the protein after For these experiments, there was no pre-incubation of enzyme with the manzamines (1 -6). For each concentration of 1 -6, five ATP concentrations varying from 0.05 to 1.2 mM were evaluated, while keeping the SA concentration constant at saturating concentrations (i.e., 5 mM SA against an apparent KM with respect to SA of 0.53 mM).…”

“…Likewise, five different concentrations of SA ranging from 0.2 to 5 mM were used for each concentration of 1 -6, while the concentration of ATP was kept constant at a saturating concentration (i.e., 1.2 mM ATP against an apparent KM with respect to ATP of 0.20 mM). 31 Each reaction was initiated by the addition of MtSK (0.2 µM), allowed to proceed for 30 seconds, and then quenched as described above.…”

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone

“…Dimethyl sulfoxide (DMSO), adenosine-5'-triphosphate (ATP), shikimate, H2O2 (30%), rabbit muscle lactate dehydrogenase (LDH) and rabbit muscle pyruvate kinase (PK) were purchased from Sigma-Aldrich (St. Louis, MO). MtSK [22] and M. tuberculosis catalase-6 peroxidase (MtKatG) [23] were expressed and purified as previously described. The purity of MtSK and MtKatG were determined by SDS-PAGE and LC-ESI-MS, and aliquots were stored at −80 °C in 50 mM Tris-HCl, pH 7.4; 0.5 M NaCl for MtSK and 5 mM phosphate, pH 7.0, for MtKatG.…”

Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate

“…In addition to being equally instable as AroL from E. coli , AroK from E. coli has been found not only with the disadvantage of a much lower shikimate affinity but also with a weaker shikimate kinase activity ( k cat not available) . Less favorable k cat / K m values were found for the shikimate kinase from Mycobacterium tuberculosis , which despite being encoded by aroK behaves more like an aroL ‐encoded shikimate kinase from its kinetic properties (from 0.9 × 10 5 M −1 s −1 for shikimate and 5.4 × 10 5 M −1 s −1 for MgATP by Rosado et al to 1.1 × 10 5 M −1 s −1 for shikimate and 5.3 × 10 5 M −1 s −1 for MgATP by Gu et al, determined by UV‐assays, while LC‐MS assays by Simithy et al gave k cat / K m values of 1.2 × 10 5 M −1 s −1 for shikimate and 3.4 × 10 5 M −1 s −1 for MgATP), for the aroK ‐encoded wild‐type and mutant shikimate kinases from Helicobacter pylori and for the aroL ‐encoded shikimate kinase from Dickeya dadantii , which was designated formerly as Erwinia chrysanthemi (1.1 × 10 5 M −1 s −1 for shikimate and 5.6 × 10 4 M −1 s −1 for MgATP) . Less favorable is also the archeal shikimate kinase from the thermophilic methanogen Methanocaldococcus jannaschii , formerly designated as Methanococcus jannaschii , with k cat / K m values of 1.1 × 10 5 M −1 s −1 for shikimate and 5.3 × 10 5 M −1 s −1 for MgATP, which have been determined at 37 °C, but the enzyme was stable at high concentrations and lost activity only at concentration below 0.01 mg mL −1 .…”

“…The 3‐phosphate group thereby contributes significantly to substrate and inhibitor recognition at the shikimic acid 3‐phosphate site of EPSPS . The shikimate kinase reaction in pathogens like M. tuberculosis , H. pylori , Acinetobacter baumannii , Acinetobacter baylyi , Haemophilus influenzae , Francisella novicida , and Pseudomonas aeruginosa is of major interest and therefore shikimic acid 3‐phosphate and shikimate kinase structure and function are also key for developing shikimate kinase inhibitors as new antimicrobials …”