Abstract
Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Biocatalysis*
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Cysteine / metabolism*
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Disulfides / metabolism
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Enzyme Stability
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Kinetics
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Models, Molecular
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Mutagenesis, Site-Directed
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Mutant Proteins / chemistry
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Mutation / genetics
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Mycobacterium tuberculosis / enzymology*
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Phosphoenolpyruvate Carboxykinase (ATP) / chemistry
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Phosphoenolpyruvate Carboxykinase (ATP) / metabolism*
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Structure-Activity Relationship
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Substrate Specificity
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Tandem Mass Spectrometry
Substances
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Disulfides
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Mutant Proteins
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Phosphoenolpyruvate Carboxykinase (ATP)
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Cysteine
Grants and funding
This work was supported by NPU project LO1302 from the Ministry of Education of the Czech Republic and by project RVO61388963. ML was supported by the Czech Science Foundation (grant number P208/12/G016). This work was supported by NPU project LO1302 from the Ministry of Education of the Czech Republic and by project RVO61388963. ML was supported by the Czech Science Foundation (grant number P208/12/G016). This work was supported from the Large Infrastructures for Research, Experimental Development and Innovations project “IT4Innovations National Supercomputing Center – LM2015070”.