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16.3: Regulation of the Citric Acid Cycle

  • Page ID
    15020
  • Entry of pyruvate into the the citric acid cycle leading to aerobic production of energy and intermediates for biosynthesis is a key metabolic step.  Hence both the pyruvate dehydrogenase complex and key enzymes in the cycle are targets for regulation.  This occurs through substrate availability, product inhibtion, allosteric effectors and post-translational modifications of key enzymes in the pathway.  A summary figure showing key regulators is shown below.

     

    TCA_WedgeDashViewReg2.svg

     

    Renee LeClair

    Regulation of the pyruvate dehydrogenase  complex (PDC) 


    Under aerobic conditions, the pyruvate produced by glycolysis will be oxidized  to acetyl CoA using the pyruvate dehydrogenase complex (PDC) in the mitochondria (note: its genes are encoded in the nucleus).  As this enzyme is a key transition point (the gatekeeper) between cytosolic and mitochondrial  metabolism  and is highly exergonic (ΔG0' = -7.9 kcal/mol), it is highly regulated by both covalent and allosteric  regulation. Deficiencies  of the PDC are X-linked and present  with symptoms of lactic acidosis  after consuming a meal  high in carbohydrates. This metabolic  deficiency can be overcome  by delivering a ketogenic diet and  bypassing glycolysis all together. 

    The PDC is regulated by allosteric and covalent regulations. The  complex itself can be allosterically  activated by pyruvate and  NAD+ Elevation of substrate (pyruvate)  will enhance flux through  this enzyme as will the indication of low energy states as triggered by  high NAD+ levels. The PDC is also inhibited by acetyl CoA and NADH directly.  Product inhibition is a very common regulatory mechanisms and  high NADH would signal sufficient energy levels, therefore decreasing activity of the PDC. The figure below summarizes the regulation.   (Adapted from Marks’ Medical Biochemistry)

     

    clipboard_e45a0c3348d004361a9075caee9c59111.png

     

    creative commons

    The PDC is also regulated through covalent modification. Phosphorylation  of the E1 subunits of the complex will decrease activity of the enzyme. 

    The enzyme responsible for phosphorylation of the PDC is pyruvate dehydrogenase  kinase. The kinase is regulated inversely to the PDC (Figure  4.9). The kinase is most active when acetylCoA, NADH and ATP are high.  These compounds will stimulate the kinase to phosphorylate and inactivate  the PDC. PDK is inhibited by dichloracetate, TPP, Ca2+, and pyruvate.   The PDC can be dephosphorylated by a calcium mediated  phosphatase, PDP.   Starvation and diabetes result in increased phosphorylation and inhibition of the complex, which impairs glucose oxidation.  

    Phosphorylation occurs on Serine 264 of the α subunit (site 1), Ser271 (site 2) and Ser203 (site 3)ar e located on a conserved phosphorylation loops. Sites 1 and 2 (in loop A) are involved in stabilization of TPP in the active site, while Ser 203 in the adjacent loop B  binds Mg2+ which stabilizes PP on bound TPP.  All it akes for inhibition is the phosphorylation of just one of the Ser side chains.  Phosphorylation prevents the ordering of the loop which occurs on TPP binding which hinders the biding of the lipoyl domains of the PDC core to E1p, which inhibits the flow of metabolites in the PDC.  

    Prevention of PDC phosphorylation by the specific PDK inhibitor dichloroacetate increases levels of reactive oxygen species in mitochondria, which promotes the expression of a mitochondria-K+ channel axis, leading to cellular apoptosis and the inhibition of tumor growth

    A summary of pathway regulation is shown below. 

    RegulationPDHComplexCC.jpg

    Legend:  

    "In general, PDC is activated through its substrates CoASH and NAD+ and kinase inhibition or phosphatase activation (PDP) (dichloroacetate, TPP, Ca2 + and pyruvate), is inhibited by its products acetyl CoA and NADH and activation of kinase (PDK). Abbreviations : PDC: pyruvate dehydrogenase complex, PDK: pyruvatedehydrogenase kinase, PDP: pyruvate dehydrogenase phosphatase TPP: thiamine pyrophosphate."

    Nasiri A, Sadeghi M, Vaisi-Raygani A, Kiani S, Aghelan Z, Khodarahmi R. Emerging regulatory roles of mitochondrial sirtuins on pyruvate dehydrogenase complex and the related metabolic diseases: Review. Biomed. Res. Ther.; 7(2):3645-3658. http://www.bmrat.org/index.php/BMRAT/article/view/591 

    The complex is also acetylated and succinylated.  

    Summary Table: Renee LeClair

    Metabolic Pathway Major Regulatory Enzyme(s) Allosteric Effectors Post-translational modifications Hormonal Effects
    Glycolysis hexokinase; glucokinase (liver) Glucose 6P (-)    
    PFK-1

    Fructose 2,6BP, AMP (+) 

    Citrate (-)

      ↑ Insulin/Glucagon leads to dephosphorylation of PFK2 and increases production of F2,6BP
    Pyruvate Kinase

    Fructose 1,6BP (+)

    ATP, Alanine (-)

      ↑ Insulin/Glucagon leads to dephosporylation
    Pyruvate Dehydrogenase PDC

    Pyruvate, NAD+ (+)

    Acetyl CoA, NADH, ATP (-)

    dephosphorylation by PDP (+)

    phosphorylation by PDK (-)

    ↑ Insulin/Glucagon leads to dephosphorylation

     

    Regulation of Key Enzymes in the Citric Acid Cycle

    Citrate synthase (ΔGo = -7.5 kcal/mol),  Isocitrate dehydrogenase (ΔGo = -2.0 kcal/mol) and alpha-ketogluatate dehydrogenase (ΔGo = -7.2 kcal/mol) are all exergonic and likely candidates for regulation.  Indeed they are. 

     

    Citrate Synthase

    Protopedia regulation of Citrate Synthase

    Isocitrate Dehydrogenase

    In the previous section, structures of the αγ and αβ heterodimer building blocks of the protein were described.  The α subunits contained the catalytic side while the β and γ submits were the regulatory subunits which bind allosteric effectors.  Crystal structure of the human αγ dimer have elucidated the mechanism of activation of the enzyme by the allosteric activators citrate and ADP.

    Following from https://www.nature.com/articles/srep40921  Creative Commons 

    Ma, T., Peng, Y., Huang, W. et al. Molecular mechanism of the allosteric regulation of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase. Sci Rep 7, 40921 (2017). https://doi.org/10.1038/srep40921

    In the absence of any allosteric activators, the active site adopts an inactive conformation unfavorable for binding of the substrate isocitrate (ICT), and the enzyme is in the basal state which is characterized by high effective Km (higher substrate concentration required for half maximal velocity) and a low catalytic efficiency. The binding of citrate (CIT, an allosteric regulator) induces conformational changes at the allosteric site, which are transmitted to the active site through conformational changes at the heterodimer interface (in both the α and γ subunits), leading to the conversion of the active site from the inactive conformation to the active conformation favorable for the ICT binding. Hence, the enzyme assumes the partially activated state which has a moderately decreased Km effective with a moderately increased catalytic efficiency. The binding of ADP in the presence of CIT does not induce further conformational changes at the allosteric site and the active site, but establishes a more extensive hydrogen-bonding network among CIT, ADP and the surrounding residues through the metal ion, which conversely enhances or stabilizes the CIT binding. Hence, the binding of CIT and ADP together has a synergistic activation effect, and the enzyme assumes the fully activated state which has a substantially decreased Km  effective with a significantly increased catalytic efficiency.

     

    IDH_AllostericReg_CIT_ADP.png

     

     

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