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Citric Acid Cycle Reactions
TCA cycle purpose, steps, regulations, reactants and products
Undergraduate 3

Additional Biochemistry Flashcards




Purpose and Basics of Citric Acid Cycle
  • Oxidize acetyl-CoA
  • the 4 and 5C intermediates are used elsewhere for biosynthesis
  • cell uses anaplerotic (replenishing) rxns to replace these intermediates
  • [image]
  • [image]
Overview of Steps
  • acetyl-CoA donates acetyl group to oxaloacetate to begin cycle
  • this forms 6C citrate
  • which becomes isocitrate
  • dehydrogenated, lose CO2, forming alpha ketoglutarate
  • lose another CO2=succinate
  • succinate then converted over three steps back into oxaloacetate
Reactions of the citric acid cycle
Step 1: Formation of Citrate
  • Condensation of oxaloacetate with acety-CoA
  • the hydrolysis of a high energy thiol intermediate makes rxn exergonic
  • large - delta G is essential b/c concentration of oxaloacetate is very low
  • CoA recycled back to PDH
  • (picture of citrate formation)
Step 1 mechanism: Citrate synthase
  • claisen condensation involving a thioester (acetylcoa) and ketone(oxaloacetate)
  • (step 1 mechanism picture)
  • oxaloacetate binds first (specifically oriented in the active site) to citrte synthase and opens binding site for acetyl coa

Step 2: citrate->cis-aconitate->isocitrate


  • (add picture of step 2)
  • aconitase can reversibly add water to ezyme bound cis aconitate, leading to citrate and iso citrate
  • though small amount of isocitrate  at equilibrium, rxn is pulled to the right because isocitrate is rapidly consumed, lowering its steady state concentration
Step 3:Oxidation of Isocitrate to alpha ketoglutarate and CO2
  • (add picture of step 3)
  • Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase forms alphaketoglutarate
  • oxalosuccinate formed in active site as intermediate
  • enol formed is stabilized by Mn++
  • NAD(P)+ rxn occurs in mitochondrial matrix and cytosol and creates NAD(P)H, used for anabolism

Step 4: Oxidation of alpha-ketoglutarate to succinyl-CoA and CO2


  • (add picture of step 4)
  • another oxidative decarboxylation
  • NAD+ is electron acceptor
  • CoA is carrier of succinyl group
  • oxidation energy of alpha-ketoglutarate conserved in forming thioester bond of succinyl CoA (and large -delta G)
  • alpha-ketoglutarate dehydrogenase complex is similar to PDH complex: has E1, E2, E3 and TPP, lipoate, FAD, NAD and CoA. differs in that it binds a-ketoglutarate, not pyruvate
Step 5: Succinyl-CoA-> Succinate
  • (add step 5 pic)
  • Succinyl coa has a thioester bond and the  negative std free energy of hydrolysis isused to drive synthesis of phosphoanhydride bond of ATP or GTP, resulting in -2.9 kJ/mol after coupling
  • energy conserving reaction where His residue is phosphorylated in active site. this P is xfered to ADP/GDP->ATP/GTP
  • formation of GTP/ATP at the expense of free energy released by a-ketoglutarate decarboxylation is a subtrate level phosphoryation
  • GTP formed can ->ATP:    GTP+ADP-->GDP + ATP  delta G=0kJ/mol
Step 6: Oxidation of Succinate to Fumarate
  • (add picture of step 6)
  • enzyme succinate dehydrogenase is tightly bound to mitochondrial inner matrix (in euks)
  • electrons pass from succinate through FAD to iron (now we're mentioning  oxidative phosphorylation) sulfur centers and then to chain of carriers in inner mitochondria membrane
  • O2 is the final electon acceptor and generates 1.5 ATP/electron pair
Step 7: Hydration of Fumarate to Malate
    • (Show step 7 picture)
  • Reversible rxn catalyzed by fumarase, has carbanion T state
  • fumarase is highly sterospecific and only binds the trans double bond of fumarate, not the cis (this isomer is called maleate)
Step 8: Oxidation of Malate to Oxaloacetate
  • (step 8 picture)
  • NAD linked L-malate dehydrogenase catalyzes the rxn of L-malate to oxaloacetate
  • under standard conditions reaction lies far to the left but within cells reaction goes right b/c oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (step1)
  • this means [oxaloacetate] in cells is very low, forcing a favorability of oxaloacetate formation
Step 5 mechanism: succinyl-CoA synthetase reaction
(add picture)
Carbon tracking; labeling acetate with C14 to determine position of isotopic carbon
  • condensation of oxalo. + acetate expected to produce a citrate labeled in one of the two carboxyl groups because citrate is a symmetrical molecule
  • half labled citrate molecules are expected to yeild a-ketoglutarate labeled in the gamma carboxyl group and half in alpha carboxyl group
  • instead, only gamma is labeled, why? because enzyme it reacts with is assymetric
  • this results in binding to enz. at three points in active site. This induced chirality of citrate due to enz. interation makes citrate prochiral
Energy oxidations in the cycle: energy conservation
  • (add energy conservation picture)
  • two carbon acetyl group combines with oxaloacetate
  • oxidation of isocitrate and a-ketoglutarate yeilds CO2
  • oxidation energy conserved in reduction of 3 NAD+ and one FAD and production of one ATP/GTP
  • four oxidation steps provide flow of electons to respiratory chain via NADH and FADH2
Citric Acid Cycle is Amphibolic
  • Meaning it can serve in both catabolism and anabolism

(show massive anabolic pathway)

  • plays a role in oxidative catabolism of fatty acids, carbs, and a. acids
  • a-ketoglutarate is precursor to asp and glu (transamination)
  • oxaloacetate-->glucose in gluconeogenesis
  • succinyl coa-->porphyrin ring in heme grps
Anapleurotic reactions replenish Citric Acid Cycle Intermediates
  • as intermediates of the cycle are removed they are replenished by anapleurotic rxns
  • (see figure on biosynthetic intermediate card; red arrows are anapleurotic)
  • ex) reversible carboxylation of pyruvate to CO2 to make oxaloacetate. when cycle is defficient in oxalo.(or other intermediates), pyruvate is carboxylated to form more oxaloacetate
Biotin in pyruvate carboxylase carries CO2 groups- description and mechanism
  • Pyruvate carboxylase reaction requires vitamin biotin, which carries carbons in their most oxidized form, CO2
  • carboxyl groups are activated in a  reaction that consumes ATP and joins CO2 to enzme bound biotin= activated CO2
  • activated CO2 is then passed to acceptor (like pyruvate) in a carboxylation reaction
  • pyruvate carboxyase has 4 subunits each containing biotin attached to a lys residue in the active site
  • pyruvate carboxylation has two steps: first a carboxyl group derived from HCO3- is attached to biotin. Next the carboxyl group is transferred to pyruvate to form oxaloacetate.
  • arm of biotin transfers activated carboxyl group from first active site to second (where ea/ step happens)
  • lipoate, biotin, and pantothenate all become covalently attached to proteins by similar reactions and all can channel substrates (like biotin)
  • (add pyruvate carboxylase mechanism)
Regulation of Citric Acid Cycle overview
  • carbon flow into citric acid cycle is regulated at the conversion of pyruvate to acetyl-CoA
  • also regulated at the entry of acetyl-CoA into the cycle
  • availability of intermediates like fatty acids used for acetyl-CoA synthesis
  • also by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase rxns
Production of Acetyl-CoA by PDH complex is regulated Allosterically and Covalently...First at the PDH complex
  • PDH is inhibited by ATP, acetyl-CoA, and NADH products. allosteric inhibition enhanced when long chain fatty acids are available (b/c--->acetyl-CoA)
  • AMP, NAD+ and CoA activate PDH (accumulate when too little acetate flows into the cycle)
  • also regulated by reversibly phosphorylateing on E1 ser residue (by two proteins)
  • the kinase is allosterically activated by high [ATP] and E1 is phosphorylated, causing PDH rxn velocity to decline
  • when [ATP] goes down, kinase activity goes down and the phosphatase removes the phosphoryl group from E1
Citric Acid Cycle is regulated at its Three Exergonic Steps
  • Three factors regulate the rate of flux through the cycle: substrate availability, product inhibition, and allosteric feedback inhibition of enzymes that catalyze early steps of the cycle
  • (show 16-18 picture)
  • the strongly exergonic steps catalyzed by citrate synthase,isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase can become rate limiting
  • availability of oxaloacetate and acetyl-CoA for citrate synthase varies with rate of citrate formation
  • NADH, product of isocitrate and a-ketoglutarate oxidation and when [NADH]/[NAD+] is high, both rxns are inhibited
  • succinyl-CoA inhibits a-ketoglutarate dehydrogenase+citrate synthase
  • citrate blocks citrate synthase
  • end product ATP blocks isocitrate dehydrogenase and citrate synthase
Citric Acid Cycle is regulated at its three exergonic steps: Activators
  • ADP allosterically activates citrate synthase
  • in muscle, calcium (signal for contraction and increase in ATP) activates isocitrate dehydrogenase and a-ketoglutarate dehydrogenase as well as the PDH complex
  • CAtalyze condensation reaction in which no nucleotide triphosphate (ATP/GTP) is required as an energy source
  • Catalyze the condensations that do use ATP or another nucleotide triphosphate energy for the synthetic reaction
Caralyze the condensation reactions in which two atoms are joined using an energy source like ATP
catalyze cleavages, or in the reverse direction additions, where electronics rearrangements occur. The PDH complex which oxidatively cleaves CO2 from pyruvate is a lyase

Transfer a phosphoryl group from a nucleotide triphosphate to an acceptor molecule. this is a phosphorylation.


Phosphorylysis is a displacement reaction in which phosphate is the attacking species and is covalently attached at the point of bond breakage
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