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
• Amino Acid Motifs MeSH
• Biocatalysis * MeSH
• Cysteine metabolism   MeSH
• Disulfides metabolism   MeSH
• Phosphoenolpyruvate Carboxykinase (ATP) chemistry   metabolism   MeSH
• Kinetics MeSH
• Models, Molecular MeSH
• Mutation genetics   MeSH
• Mutagenesis, Site-Directed MeSH
• Mutant Proteins chemistry   MeSH
• Mycobacterium tuberculosis enzymology   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Enzyme Stability MeSH
• Substrate Specificity MeSH
• Tandem Mass Spectrometry MeSH
• Protein Structure, Tertiary MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cysteine MeSH
• Disulfides MeSH
• Phosphoenolpyruvate Carboxykinase (ATP) MeSH
• Mutant Proteins MeSH