INTRODUCTION

        The Krebs cycle is the primary site of the
     oxidation of carbon atoms derived from food.
 

        It is sometimes called the "final common
            pathway" because all carbon atoms
                from food are oxidized here.

OVERVIEW OF THE KREBS CYCLE:

    AKA tricarboxylic acid cycle, citric acid cycle.

    Major function is the oxidation of carbon atoms.

    All reactions of the Krebs cycle occur in the
                        mitochondrion.

    Net Stoichiometry:

  Acetyl CoA + 3NAD⁺ + FAD + GDP + P_i +H₂O

                               <=====>

   2CO₂ + 3 NADH  +  FADH₂ + CoA-SH + GTP
                   + 3H⁺

        The Acetyl CoA is made from pyruvate by
                    pyruvate dehydrogenase.

REACTIONS OF THE KREBS CYCLE

    1) Formation of citrate:

         Acetyl CoA +          à     Citrate +
     Oxaloacetate (OAA)            CoA-SH

        Catalyzed by: Citrate synthase

    The hydrolysis of the thiolester of Acetyl CoA
            releases energy used to form citrate.

    Citrate is a tri-carboxylic acid, thus the name
      ‘tricarboxylic acid cycle’ (TCA Cycle).

     Being the first enzyme in the pathway, citrate
        synthase is a major regulatory enzyme
                            (more later).

   2) Isomerization:

        Citrate à   {cis-Aconitate} à   Isocitrate
                             enzyme bound
                              intermediate

        Both reactions catalyzed by: Aconitase

    3) Decarboxylation:

    Isocitrate + NAD⁺ àα-keto glutarate +
                                     CO₂ + NADH + H⁺

        Catalyzed by: Isocitrate dehydrogenase (dh).

        This is also a major regulatory enzyme
                        (more later).

    4) Formation of thiolester:

        α-Keto Glutarate +   à   Succinyl-CoA + CO₂
          CoA-SH + NAD⁺                 NADH + H⁺

    Catalyzed by: α-Keto Glutarate Dehydrogenase

    This reaction is analogous to the Pyruvate dh
                            reaction:

        It has the same three parts.

        It has the same five cofactors.

    This is the third important regulatory step in
            the Krebs cycle (more later).

    5) Formation of GTP:

        Succinyl-CoA +   à    Succinate + GTP +
            GDP + P_i                     CoA-SH

            Catalyzed by Succinic Thiokinase
                (Succinyl-CoA Synthetase).

    The hydrolysis of the thiolester of Succinyl-
  CoA yields the energy needed to produce GTP:

       This is another example of substrate level
                    phosphorylation.

    6) FAD-linked oxidation of succinate:

        Succinate + FAD à Fumarate + FADH₂

            Catalyzed by: Succinic Dehydrogenase

                This enzyme is bound to the inner
                 mitochondrial membrane, and is
                   actually part of the electron
                       transport system (ETS).

                Thus, the electrons on the FADH₂
                  can enter the ETS directly, and
                       their energy can be used
                              to produce ATP.

            This is a fourth regulatory step in the
                     Krebs cycle (more later).

    7) Hydration:

            Fumarate + H₂O à Malate

                Catalyzed by: Fumarase

    8) NAD+-linked oxidation of malate:

        Malate + NAD⁺ à Oxaloacetate +
                                         NADH + H⁺

            Catalyzed by: Malate dehydrogenase.

            This reaction forms oxaloacetate (OAA),
                     one of the precursors, so this
                           pathway is a cycle!

INPUT/OUTPUT RELATIONSHIPS OF THE
                    KREBS CYCLE

        Input                            Output

   Acetyl CoA                           2 CO₂

                                                 CoA-SH

   3 NAD⁺                                3 NADH + 3 H⁺

   1 FAD                                   1 FADH₂

   GDP + P_i                                      GTP

    Note: No ATP is produced in the Krebs cycle!

       However, Using the nucleotide kinase reaction,
            the GTP can be used to produce ATP:

                 GTP + ADP ßà ATP + GDP

REGULATION OF THE KREBS CYCLE

    Overview:

        The cycle is regulated by the cell’s need for
                both Energy and Carbon.

        Also regulated by the availability of oxygen
                            (more later).

    There are four regulatory steps in the cycle:

        Citrate Synthase:

            Inhibited by citrate, succinyl-CoA, NADH, ATP.

            Activated by ADP.

        Isocitrate Dehydrogenase:

            Inhibited by ATP and NADH

            Activated by ADP, NAD⁺, and calcium ions
                (signal for muscle contraction).

        α-keto Glutarate dehydrogenase:

            Inhibited by NADH, Succinyl CoA, ATP,
                            and GTP.

            Activated by calcium ions.

        Succinate Dehydrogenase:

            Inhibited by OAA.

            Activated by Succinate, ADP, and P_i.

    The Krebs cycle and Glycolysis are regulated in
      tandem, so that under most conditions, only as
       much pyruvate and acetyl CoA are produced
          from glycolysis as are needed to supply
                        the Krebs cycle.

    This is accomplished by the feedback inhibition
     of PFK by ATP and NADH, which are signals
    of the energy supply, and by feedback inhibition
    by citrate, which is a signal of the substrate needs.

AMPHIBOLIC NATURE OF THE TCA CYCLE

    The Krebs cycle has both catabolic and anabolic
            functions, and therefore is amphibolic.

    Catabolic functions:

        Oxidation of Acetyl CoA and other inter-
          mediates derived from other pathways:

            α-Keto Glutarate, Succinyl CoA, OAA.

        Formation of high energy intermediates:

            NADH + H⁺, FADH2, and GTP.

    Anabolic functions:

        Use of Krebs cycle intermediates to make
                            other things:

            Citrate: part of fatty acid synthesis pathway.

            OAA: part of gluconeogenesis and
                            amino acid synthesis.

            α-KG: part of amino acid synthesis.

            Succinyl CoA: part of heme and
                    chlorophyll synthesis.

ANAPLEROTIC REACTIONS OF TCA CYCLE

    Due to the amphibolic nature of the cycle,
    sometimes the supply of intermediates can
                    become limiting.

    Remember than when we add 2 carbons
     as Acetyl CoA, 2 carbons come out as
  CO₂, and thus we can’t add to the carbon
  pool of the Krebs cycle with Acetyl CoA.

    A corollary to this is that we can’t make
           glucose from Acetyl-groups.

    There are three reactions that can add carbon
                    to the Krebs cycle:
            (‘anaplerotic’ means ‘to fill up’)

      1. Pyruvate carboxylase (liver and kidney):

    Pyruvate + CO₂ + ATP ßà OAA + ADP + P_i
        (3C)                                    (4C)

    This enzyme is allosterically activated by
                        Acetyl CoA.

    Thus, when the supply of Acetyl CoA is
      high, more OAA is made so that it can
   enter the cycle(this will be important later).

    As with most carboxylases, this enzyme
            requires biotin as a cofactor.

    2. Phosphoenolpyruvate carboxykinase (PEPCK)
            (heart and skeletal muscle):

        PEP + CO₂ + GDP ßà OAA + GTP

    3. Malic enzyme (widely distributed-cytosolic
                        in mammals):

    Pyruvate + NADPH + H⁺ ßà Malate + NADP⁺
                + CO₂