Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

• Klíčová slova
• Mycobacterium tuberculosis, allosteric regulation, enzyme kinetics, glycolysis, phosphofructokinase A and B,
• MeSH
• adenosindifosfát metabolismus   farmakologie   MeSH
• adenosintrifosfát metabolismus   farmakologie   MeSH
• alosterická regulace MeSH
• bakteriální proteiny antagonisté a inhibitory   metabolismus   MeSH
• enzymová indukce MeSH
• fosfofruktokinasy antagonisté a inhibitory   metabolismus   MeSH
• fruktosadifosfáty biosyntéza   farmakologie   MeSH
• fruktosafosfáty metabolismus   farmakologie   MeSH
• glukoneogeneze MeSH
• glykolýza MeSH
• hexosafosfáty metabolismus   MeSH
• izoenzymy antagonisté a inhibitory   metabolismus   MeSH
• katalýza MeSH
• kinetika MeSH
• kyslík farmakologie   MeSH
• L-laktátdehydrogenasa metabolismus   MeSH
• Mycobacterium tuberculosis účinky léků   enzymologie   MeSH
• pyruvátkinasa metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• substrátová specifita MeSH
• zpětná vazba fyziologická MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• bakteriální proteiny MeSH
• fosfofruktokinasy MeSH
• fructose-1,6-diphosphate MeSH Prohlížeč
• fructose-6-phosphate MeSH Prohlížeč
• fruktosadifosfáty MeSH
• fruktosafosfáty MeSH
• hexosafosfáty MeSH
• izoenzymy MeSH
• kyslík MeSH
• L-laktátdehydrogenasa MeSH
• phosphofructokinase A protein, Mycobacterium tuberculosis MeSH Prohlížeč
• pyruvátkinasa MeSH
• rekombinantní proteiny MeSH
• tagatose 6-phosphate MeSH Prohlížeč

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
• aminokyselinové motivy MeSH
• biokatalýza * MeSH
• cystein metabolismus   MeSH
• disulfidy metabolismus   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie   metabolismus   MeSH
• kinetika MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   MeSH
• Mycobacterium tuberculosis enzymologie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• disulfidy MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• mutantní proteiny MeSH

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

• MeSH
• aktivace enzymů MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie   genetika   metabolismus   MeSH
• glukoneogeneze MeSH
• katalýza MeSH
• kationty dvojmocné MeSH
• kineze MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• Mycobacterium tuberculosis enzymologie   genetika   MeSH
• nukleotidy metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• kationty dvojmocné MeSH
• nukleotidy MeSH