Citric Acid Cycle

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The tricarboxylic acid cycle

  • Overview of the tricarboxylic acid(TCA)cycle(Krebs cycle,citric acid cycle)
    • Involves the oxidation of acetyl coenzyme A (CoA) along with the reduction of coenzymes, which are subsequently reoxidized to produce ATP
    • The cycle isamphibolic, providing carbon skeletons for (MCQ)
      • Gluconeogenesis
      • fatty acid synthesis
      • interconversion of amino acids.
    • All the enzymes of the TCA cycle are in the mitochondrial matrixexcept succinate dehydrogenase, which is in the inner mitochondrial membrane (MCQ)
  • Entry of pyruvate from glycolysis
    • In order for carbons from glucose to enter the TCA cycle, glucose is first converted to pyruvate by glycolysis, then pyruvate forms acetyl CoA.
    • Reactionsequence
    • Pyruvate dehydrogenase (PDH)
      • amultienzyme complex
      • locatedexclusively in themitochondrial matrix(MCQ)
      • catalyzes the oxidative decarboxylation of pyruvate, formingacetyl CoA, carbon dioxide, and NADH.
      • The reactions catalyzed by the pyruvate dehydrogenase complex (PDHC) are analogous to those catalyzed by the alpha-ketoglutarate dehydrogenase complex
  • Regulation of pyruvate dehydrogenase(PDH)
    • In contrast to a-ketoglutarate dehydrogenase, PDH exists in a phosphorylated

      (inactive) form and a dephosphorylated (active) form.(MCQ)

      • A kinaseassociated with the multienzyme complex phosphorylates the pyruvatedecarboxylase subunit, inactivating the PDHC.
      • TheproductsofthePDHreaction,acetylCoAandNADH,activatethekinase(MCQ)
      • Thesubstrates,CoAandNAD+,inactivatethekinase.
      • The kinase is also inactivated by ADP(MCQ)
      • AphosphatasedephosphorylatesandactivatesthePDHC.(MCQ)
      • When the concentration of substrates is high, the dehydrogenase is active, andpyruvate is converted to acetyl CoA. (MCQ)
      • When the concentration of products is high, the dehydrogenase is relatively inactive.(MCQ)
    • The reactions of the TCA cycle
      • Acetyl CoA and oxaloacetate (OAA) condense, forming citrate.
        • Enzyme:citratesynthase.
        • Cleavage of the high-energy thioester bond in acetyl CoA provides the energy for this condensation.
        • Citrate(theproduct)isaninhibitorofthisreaction.(MCQ)
      • Citrate is isomerized to isocitrateby a rearrangement of the molecule
        • Enzyme:aconitase.
        • Aconitateservesasanenzyme-boundintermediate.
      • Isocitrateis oxidized to a-ketoglutarate
        • firstoxidative decarboxylation reaction(MCQ)
        • CO2 is produced
        • electrons are passed to NAD+ to form NADH + H+.
        • Enzyme:isocitratedehydrogenase.
        • This key regulatory enzyme of the TCA cycle is allostericallyactivated by ADP andinhibited by NADH.(MCQ)
      • alpha-Ketoglutarateis converted to succinyl CoA
        • secondoxidative decarboxylation reaction.
        • CO2 is released
        • succinyl CoA, NADH, and H+ are produced.
        • Enzyme:alpha-ketoglutaratedehydrogenase.
        • ThisenzymerequiresthesamefivecofactorsasdoesPDH:thiaminepyrophosphate,lipoicacid, uncombined coenzyme A (CoASH), flavin adenine dinucleotide (FAD), and NAD+.
      • Succinyl CoA is cleaved to succinate.
        • Cleavage of the high-energy thioester bond of succinyl CoA
        • provides energy for the substrate level phosphorylationof guanosinediphosphate (GDP) to guanosine triphosphate (GTP). (MCQ)
        • Because this does not involve the electron transport chain, it is not an oxidative phosphorylation.
        • Enzyme:succinatethiokinase.
        • The enzyme is also called succinyl CoA synthetase.
      • Succinate is oxidized tofumarate.
        • Succinate transfers two protons together with their electronsto FAD, which forms FADH2.
        • Enzyme:succinatedehydrogenase.
        • This enzyme is present in the inner mitochondrial membrane. (MCQ)
        • The other enzymes ofthe cycle are in the matrix.
      • Fumarate is converted to malate
        • Done by the addition of water across the double bond.
        • enzymefumarase
      • Malate is oxidized regenerating OAA
        • enzymemalate dehydrogenase, and
        • Two protons along with their electrons are passed to NAD+, producing NADH + H+.(MCQ)
    • Energy production by theTCA cycle
      • The NADH and FADH2 (produced by the cycle) donate electrons to the electron transport chain.
      • For each mole of NADH, about 2.5 moles of ATP are generated(MCQ)
      • For each mole of FADH2, about 1.5 moles of ATP are generated by the passage of these electrons to O2 (oxidative phosphorylation). (MCQ)
      • In addition, GTP is produced when succinyl CoA is cleaved.
        • GTP produces ATP.
      • The total energy generated by one round of the cycle, starting with one mole of acetyl CoA, is about 10 moles of ATP (MCQ)
    • Regulation of the TCA cycle
      • ADP allosterically activatesisocitrate dehydrogenase.
        • The TCA cycle is regulated by the cell’s need for energyin the form of ATP
        • When ATP is utilized, ADP and inorganic phosphate (Pi) are produced.
        • When ADP levels are high relative to ATP—that is, when the cell needs energy—the reactions of the electron transport chain are accelerated.
        • NADHisrapidlyoxidized;consequently,theTCAcyclespeedsup.(MCQ)
      • NADH allosterically inhibits isocitrate dehydrogenase.(MCQ)
        • When the concentration of ATP is high—the cell has an adequate energy supplythe electron transport chain slows down
        • NADH builds up,  consequently, the TCA cycle is inhibited.
      • Citrate inhibits citrate synthase, the first enzyme of the cycle
      • High NADH (low NAD+) levelsslows the cycle by mass action.
        • OAA is converted to malate when NADH is high(MCQ)
        • therefore, less substrate is available for the citrate synthase reaction.
    • Co factors and vitamins required for reactions of the TCA cycle
      • NAD+ accepts a hydride ion, which reacts with its nicotinamide ring
        • The nicotinamide ring of NAD+ is derived from the vitamin niacin (nicotinic acid) and, to a limited extent, from the amino acid tryptophan.(MCQ)
        • NAD+ is used in the isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and malate dehydrogenase reactions, as well as the PDH reaction.
      • FAD accepts two hydrogen atoms (with their electrons).
        • FAD is derived from thevitamin riboflavin.
        • FAD is the cofactor for succinate dehydrogenase(MCQ)
        • FAD is also required by alpha-ketoglutaratedehydrogenase and PDH.(MCQ)
      • Coenzyme A
        • CoA contains a sulfhydryl group that reacts with carboxylic acids to form thioesters, CoAcontainsthevitaminpantothenicacid.(MCQ)
        • CoAisusedinthealpha-ketoglutaratedehydrogenaseandPDHcomplex.
      • Thiamineandlipoicacid,cofactorsfora-ketoaciddehydrogenases(MCQ)
        • a-Ketoacid dehydrogenases catalyze oxidative decarboxylations
          • PDH, oxidativelydecarboxylates pyruvate, forming acetyl CoA
          • a-Ketoglutarate dehydrogenase, catalyzes the conversion of a-ketoglutarate tosuccinyl CoA
    • Synthetic functions of the TCA cycle
      • Intermediates of the TCA cycle are used in the(MCQ)
        • fasting state in the liver for the production ofglucose
        • in the fed state for the synthesis of fatty acids.
        • used to synthesize amino acids
        • to convert one amino acid to another.
      • Anaplerotic reactionsreplenish intermediates of the TCA cycle as they are removed for the synthesis of glucose, fatty acids, amino acids, or other compounds.
        • A key anaplerotic reaction is catalyzed by pyruvate carboxylase(MCQ)
        • carboxylates pyruvate, forming OAA. (Step 1)
          • Pyruvate carboxylase requires biotin, a cofactor that is commonly involved in CO2 fixation reactions.
          • Pyruvate carboxylase, found in liver, brain, and adipose tissue (but not in muscle),
          • Pyruvate carboxylase,isactivated by acetyl CoA.
        • Amino acids produce intermediates of the TCA cycle through anaplerotic reactions.(MCQ)
          • Glutamate is converted to a-ketoglutarate. (MCQ)
            • Amino acids that form glutamateinclude glutamine, proline, arginine, and histidine.
          • Aspartate is transaminated to form OAA. (MCQ)
            • Asparagine can produce aspartate.
          • Valine, isoleucine, methionine, and threonine produce propionylCoA(MCQ)
          • propionyl CoA is converted to methylmalonyl CoA and, subsequently, to succinyl CoA, an intermediate ofthe TCA cycle.
          • Phenylalanine, tyrosine, and aspartateform fumarate. (MCQ)
      • Synthesis of glucose
        • The synthesis of glucose occurs by the pathway of gluconeogenesis, which involves intermediates of the TCA cycle.
        • As glucose is synthesized, malate or OAAis removed from the TCA cycle and replenished by anaplerotic reactions.
        • Pyruvate, produced from lactate or alanine, is converted by pyruvate carboxylase to OAA, which forms malate. (MCQ)
        • Various amino acids that supply carbon for gluconeogenesisare converted to intermediates of the TCA cycle, which form malate and, thus, glucose.
      • Synthesis of fatty acids
        • The pathway for fatty acid synthesis from glucose includes reactions of the TCA cycle.
        • From glucose, pyruvate is produced and converted to OAA (by pyruvate carboxylase) and to acetyl CoA (by PDH).(MCQ)
        • OAAandacetylCoAcondensetoformcitrate,whichisusedforfattyacidsynthesis.(MCQ)
        • Pyruvate carboxylase catalyzes the anaplerotic reaction that replenishes the TCA cycle intermediates.
      • Synthes is of amino acids
          • Synthesis of amino acids from glucose involves intermediates of the TCA cycle.
            • Glucose is converted to pyruvate, which forms OAA, which by transamination formsaspartate and, subsequently, asparagine.(MCQ)
            • Glucose is converted to pyruvate, which forms both OAA and acetyl CoA, which con- dense, forming citrate.
              • Citrate forms isocitrate and then a-ketoglutarate, producing glutamate, glutamine, proline, and arginine.(MCQ)
          • Inter conversion of amino acids involves intermediates of the TCA cycle
            • For example, the carbons of glutamatecan feed into the TCA cycle at the a-ketoglutarate level and traverse the cycle, forming OAA, which may be transaminated toaspartate.(MCQ)

             

            Citric Acid Cycle

            1.         Pyruvate dehydrogenase complex (PDH) consists of which set of enzymes:

            A.        Pyruvate decarboxylase, dihydrolipoyltransacetylase

            B.        Dihydrolipoyl dehydrogenase, dihydrolipoyltransacetylase

            C.        Dihydrolipoyl decarboxylase, pyruvate decarboxylase, dihydrolipoyltransacetylase

            D.        Pyruvate decarboxylase, dihydrolipoyltransacetylase, dihydrolipoyl dehydrogenase

            ANS:   1.         D. Pyruvate is oxidativelydecarboxylated to acetyl CoA by pyruvate dehydrogenase complex (PDH). PDH is a multienzyme complex consisting of pyruvate decarboxylase, dihydrolipoyltransacetylase and dihydrolipoyl dehydrogenase. It is an irreversible process taking place in mitochondrial matrix.

            2.         Coenzymes required for oxidative decarboxylation of pyruvate to acetyl CoA:

            A.        Thiamine pyrophosphate (TPP), lipoic acid, flavin adenine dinucleotide (FAD)

            B.        TPP, lipoic acid, coenzyme A, FAD

            C.        TPP, lipoic acid, coenzyme A, FAD, nicotinamide adenine dinucleotide (NAD+)

            D.        Lipoic acid, coenzyme A, NAD+, FAD

            ANS:   2.         C. Pyruvate reacts with thiamine pyrophosphate (TPP) and then is decarboxylated.

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            Lipoic acid is regenerated by dihydrolipoyl dehydrogenase. This enzyme uses a bound FAD to accept reducing equivalents. The FADH2 produced by this reaction is regenerated by using NAD+.

            3.         Which one of the following statement is correct regarding regulation of PDH:

            A.        PDH is allosterically inhibited by NAD

            B.        PDH kinase catalyses the dephosphorylation and inactivation of PDH

            C.        PDH phosphatase catalyses the dephosphorylation and activation of PDH

            D.        Insulin inhibits the enzyme PDH in adipose tissue

            ANS:   3. C. Acetyl CoA and NADH compete with NAD+ and CoA for binding sites on the enzymes and allosterically inhibit the enzyme PDH. The PDH complex can exist in two forms active dephosphorylated form and inactive phosphorylated form. Two enzymes associated with PDH complex are PDH kinase and PDH phosphatase. PDH kinase is activated by the products NADH, acetyl CoA and ATP resulting in an inactivation of PDH. (PDH phosphatase dephosphorylates and activates PDH). Phosphorylation of the enzyme is inhibited by pyruvate, CoA, NAD+, AMP and ADP resulting in activation of PDH. Insulin and Ca2+activates dephosphorylated PDH phosphatase in adipose tissue, which leads to an increase in the formation of acetyl CoA.

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            4.         A patient has dietary deficiency of thiamine which results in:

            A.        Increase in the activity of PDH

            B.        Increase in the serum lactate

            C.        Decrease in serum pyruvate

            D.        Decrease in the serum lactate

            ANS:   4.B      Dietary deficiency of thiamine leads to a deficiency of the coenzyme thiamine pyrophosphate (TPP) which results in decrease in the activity of PDH and an accumulation of pyruvate. Lactate and alanine are also high because they may be formed from pyruvate by the reversible activities of lactate dehydrogenase and alanine transaminase. Lactate is an acid, high level of lactate in the blood leads to lactic acidosis. Thiamine deficiency can lead to beriberi and wernicke’s syndrome.

            5.         Succinate dehydrogenase is mainly present in:

            A.        Cytosol

            B.        Mitochondrial matrix

            C.        Inner mitochondrial membrane

            D.        All of the above

            ANS:   5.C.     All the enzymes of tricarboxylic acid (TCA) cycle present in mitochondrial matrix are soluble, but succinate dehydrogenase (SDH) is found on the inner face of the inner mitochondrial membrane. It is an example of an iron-sulphate protein. SDH enzyme is composed of two subunits with mol. wt. 70000 and 30000. The 70000 mol wt. subunit contains the substrate- binding site.

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            6.         Which one of the following is a condensation reaction in TCA cycle:

            A.        Formation of citrate

            B.        Conversion of citrate to isocitrate

            C.        Conversion of a-ketoglutarate to succinyl CoA

            D.        Formation of malate

            ANS:   6.         A. An initial step in the TCA cycle is the condensation reaction. Citrate is formed from the condensation of an acetyl CoA with oxaloacetate, this reaction is catalyzed by the enzyme citrate synthase. Citrate is isomerizes to isocitrate by the enzyme aconitase. a-ketoglutarate is oxidativelydecarboxylated to succinyl CoA by a-ketoglutarate dehydrogenase. Fumarate is converted to malate by fumarase, utilizing one water molecule.

            7.         Substrate level phosphorylation step in TCA cycle is catalyzed by:

            A.        Isocitratedehydrogenase

            B.        a-ketoglutaratedehydrogenase

            C.        SuccinylCoAsynthase

            D.        Succinatedehydrogenase

            ANS:   7.         C. Succinyl CoA is converted to succinate by succinyl CoA synthase. In this reaction GDP is phosphorylated to GTP. Nucleoside diphosphate kinase enzyme converts GTP to ATP by transferring y-phosphate from GTP to ADP. This is a substrate level phosphorylation step in TCA cycle.

            8.         How many ATPs are produced by oxidation of one acetyl CoA via TCA cycle:

            A.        9 ATP

            B.        10 ATP

            C.        13 ATP

            D.        12 ATP

            ANS:   8.         D. 12 ATPs are produced by oxidation of one acetyl CoA via TCA cycle.

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            9.         How many ATPs are produced in the conversion of pyruvate to citrate:

            A.        2

            B.        3

            C.        4

            D.        1

            ANS:   9. B.Total 3 ATPs are produced in the conversion of pyruvate to citrate.

            10.       Which one of the following key regulatory enzyme in TCA cycle isallosterically regulated by succinylCoA:

            A.        Citrate synthase

            B.        Isocitratedehydrogenase

            C.        a-Ketoglutarate dehydrogenase

            D.        Both citrate synthase and a-ketoglutarate dehydrogenase

            ANS:   10. D.Isocitrate dehydrogenase and a-ketoglutarate dehydrogenase are allosterically regulated by succinyl CoA.

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            11.       Which enzyme is inhibited by fluoroacetate:

            A.        Aconitase

            B.        a-Ketoglutarate

            C.        Succinatedehydrogenase

            D.        Citrate synthase

            ANS:   11.       A. Fluroacetate is activated to monofluroacetyl CoA. Citrate synthase reacts with monofluroacetylCoA to form mono-flurocitrate. Aconitase enzyme is inhibited by flurocitrate; this is an example for suicide inhibition. Arsenite inhibits the enzyme by binding to the SH group of lipoic acid in the oc-ketoglutarate complexes binding of these inhibitors prevents the peroxidation of the SH group of a disulfide. Malonate is a competitive inhibitor of succinate dehydrogenase.

            12.       Which intermediate of TCA cycle is involved in the synthesis of heme?

            A.        Oxaloacetate

            B.        Citrate

            C.        SuccinylCoA

            D.        oc-Ketoglutarate

            ANS:   12.       C. Succinyl CoA is essential for the synthesis of heme. Oxaloacetate and a-ketoglutarate are precursors for the synthesis of aspartate and glutamate. Citrate formed in mitochondria is transported to the cytosol. Acetyl CoA is the cleavage product of citrate which is essential for the biosynthesis of metabolic compounds (fatty acids, sterols).

            13.       Oxaloacetate in TCA cycle can be synthesized from:

            A.        oc-Ketoglutarate

            B.        Pyruvate

            C.        Citrate and pyruvate

            D.        Pyruvate and aspartate

            ANS:   13.       D. Biotin dependent pyruvate carboxylase forms oxaloacetate from pyruvate. Aspartate on transamination forms oxaloacetate. Glutamate on transamination forms a-ketoglutarate.

            1    A chronic alcoholic, while out on a binge, became very confused and forgetful. The police found the man and brought him to the emergency department. Upon examination, he displayed nystagmus and ataxia. Which enzyme is displaying reduced activity in his brain under these conditions?

            (A)       Glyceraldehyde-3-phosphate dehydrogenase

            (B)       Isocitrate dehydrogenase

            (C)       α-ketoglutarate dehydrogenase

            (D)       Succinate dehydrogenase

            (E)       Malate dehydrogenase

            1 The answer is C:

            a-ketoglutarate dehydrogenase. The alcoholic has become deficient in vitamin B1, thiamine, which is converted to thiamine pyrophosphate for use as a coenzyme. One of the symptoms of B1 deficiency is neurological, due to insufficient energy generation within the nervous system. B1 is required for a small number of enzymes, including transketolase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase. By reducing the activity of the latter two enzymes, glucose oxidation to generate energy is impaired, and the nervous system suffers because of it.

            2    The energy yield from the complete oxidation of acetyl- CoA to carbon dioxide is which of the following in terms of high-energy bonds formed?

            (A)       6

            (B)       8

            (C)       10

            (D)       12

            (E)       14

            2 The answer is C:

            10. When acetyl-CoA enters the TCA cycle, and is converted to two molecules of carbon dioxide, and oxaloacetate is regenerated, three molecules of NADH are produced, along with one molecule of FADH2 and one substrate-level phosphorylation resulting in the generation of GTP.

                  As each NADH can give rise to 2.5 ATP, and each FADH2 to 1.5 ATP via oxidative phosphorylation, the net yield of high-energy bonds from one acetyl-CoA being oxidized by the cycle is 10 (7.5 from NADH, 1.5 from FADH2, and 1 from GTP). This is shown in the figure below.

             


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