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.
- MeSH
- Adenosine Diphosphate metabolism pharmacology MeSH
- Adenosine Triphosphate metabolism pharmacology MeSH
- Allosteric Regulation MeSH
- Bacterial Proteins antagonists & inhibitors metabolism MeSH
- Enzyme Induction MeSH
- Phosphofructokinases antagonists & inhibitors metabolism MeSH
- Fructosediphosphates biosynthesis pharmacology MeSH
- Fructosephosphates metabolism pharmacology MeSH
- Gluconeogenesis MeSH
- Glycolysis MeSH
- Hexosephosphates metabolism MeSH
- Isoenzymes antagonists & inhibitors metabolism MeSH
- Catalysis MeSH
- Kinetics MeSH
- Oxygen pharmacology MeSH
- L-Lactate Dehydrogenase metabolism MeSH
- Mycobacterium tuberculosis drug effects enzymology MeSH
- Pyruvate Kinase metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Substrate Specificity MeSH
- Feedback, Physiological MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Keywords
- Prevapis Adult, E.P.I.D., MD přípravky, citikolin,
- MeSH
- Antioxidants therapeutic use MeSH
- Cytidine Diphosphate Choline administration & dosage pharmacology therapeutic use MeSH
- Phosphates metabolism MeSH
- Fructosediphosphates administration & dosage pharmacology therapeutic use MeSH
- Phytotherapy MeSH
- Physical Endurance * drug effects MeSH
- Glutathione administration & dosage pharmacology therapeutic use MeSH
- Immunization methods MeSH
- Collagen therapeutic use MeSH
- Ascorbic Acid pharmacology therapeutic use MeSH
- Humans MeSH
- Musculoskeletal Pain drug therapy MeSH
- Oxidative Stress drug effects MeSH
- Dietary Supplements MeSH
- Probiotics pharmacology therapeutic use MeSH
- Propolis pharmacology therapeutic use MeSH
- Plant Extracts MeSH
- Athletic Injuries * prevention & control MeSH
- Athletic Performance * MeSH
- Sports Medicine MeSH
- Check Tag
- Humans MeSH
- Keywords
- citikolin,
- MeSH
- Cytidine Diphosphate Choline therapeutic use MeSH
- Energy Metabolism drug effects MeSH
- Fructosediphosphates therapeutic use MeSH
- Glutathione therapeutic use MeSH
- Wound Healing drug effects MeSH
- Homeostasis * drug effects MeSH
- Infusions, Intravenous * MeSH
- Ascorbic Acid therapeutic use MeSH
- Humans MeSH
- Neuroprotective Agents MeSH
- Oxidative Stress drug effects MeSH
- Convalescence MeSH
- Vitamin B Complex therapeutic use MeSH
- Check Tag
- Humans MeSH
Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Models, Biological MeSH
- Fructosediphosphates metabolism MeSH
- Lactococcus lactis metabolism MeSH
- Metabolic Networks and Pathways MeSH
- Carbohydrate Metabolism * MeSH
- Monte Carlo Method MeSH
- Computer Simulation MeSH
- Models, Statistical MeSH
- Feedback, Physiological MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
