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Updated: Jul 30, 2025
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Published on: April 5, 2017
The Mycobacterium tuberculosis protein tyrosine phosphatase MptpA features a pH dependent activity overlapping the
Michael Kovermann1, Alessandra Stefan2, Chiara Palazzetti3
1Department of Chemistry, University of Konstanz, Universitätstraße 10, 78464, Konstanz, Germany.
Abstract:
The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium pathogenicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic conditions in vivo, we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K0.5 of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa2 equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive inhibitors bearing a negatively charged group featuring pKa values lower than that of the substrate phosphate group.
Insights
Mycobacterium tuberculosis MptpA, essential for virulence, loses catalytic efficiency and undergoes conformational changes in acidic conditions. This suggests targeting MptpA with inhibitors effective at low pH for tuberculosis treatment.
Area of Science:
- Biochemistry
- Microbiology
- Structural Biology
Background:
- Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) is crucial for pathogenicity by inhibiting phagosome-lysosome fusion.
- MptpA's function is linked to maintaining a non-acidic environment for bacterial survival within host cells.
- Previous studies focused on MptpA's properties at neutral to alkaline pH (pH 8.0).
Purpose of the Study:
- To investigate the pH-dependence of MptpA's structural and catalytic properties.
- To understand how acidic conditions affect MptpA's interaction with phosphotyrosine (pTyr).
- To evaluate the efficacy of MptpA inhibitors at acidic pH.
Main Methods:
- Analysis of MptpA's structural and catalytic properties across a pH gradient.
- Determination of kinetic parameters, including K0.5 for pTyr.
- Surface plasmon resonance (SPR) to assess MptpA-pTyr binding.
- Evaluation of inhibitor L335-M34 effectiveness at varying pH.
Main Results:
- MptpA exhibits significant conformational changes and reduced catalytic efficiency at acidic pH.
- Enzymatic activity decreases notably as pH drops below 6.5, with a pKa2 of 5.7 for the phosphotyrosine substrate.
- MptpA shows poor binding to pTyr at pH < 6.5, while inhibitor L335-M34 demonstrates enhanced efficacy at pH 6.
Conclusions:
- MptpA's function is highly sensitive to acidic pH, impacting its virulence role.
- The findings suggest a strategy for developing novel anti-tuberculosis drugs targeting MptpA.
- Competitive inhibitors with negatively charged groups and lower pKa values than the substrate phosphate are promising therapeutic candidates.
