Chapt. 20 TCA cycle

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Chapt. 20 TCA cycle

• Ch. 20 Tricarboxylic acid cyle
• Student Learning Outcomes
• Describe relevance of TCA cycle
• Acetyl CoA funnels products
• Describe reactions of TCA cycle in cell
  respiration 2C added, oxidations,
  rearrangements-gt NADH, FAD(2H), GTP, CO2 produced
• Explain TCA cycle intermediates are used in
  biosynthetic reactions
• Describe how TCA cycle is regulated by ATP
  demand ADP levels, NADH/NAD ratio

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Overview TCA cycle

• TCA cycle (Krebs cycle)
• or citric acid cycle
• Generates 2/3 of ATP
• 2C unit Acetyl CoA
• Adds to 4C oxaloacetate
• Forms 6C citrate
• Oxidations, rearrangements -gt
• Oxaloacetate again
• 2 CO2 released
• 3 NADH, 1 FAD(2H)
• 1 GTP

Fig. 1
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II. Reactions of TCA cycle

• Reactions of TCA cycle
• 2 C of Acetyl CoA are oxidized to CO2 (not the
  same 2 that enter)
• Electrons conserved through NAD, FAD -gt go to
  electron transport chain
• 1 GTP substrate level phosphorylation
• 2.5 ATP/NADH 1.5 ATP/FAD(2H)
• Net 10 high-energy P/Acetyl group

Fig. 2
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TCA cycle reactions

• TCA cycle Reactions.
• A. Formation, oxidation of isocitrate
• 2C onto oxaloacetate
• (synthase C-C
• synthetases need P)
• Aconitrase move OH
• (will become CO)
• Isocitrate Dehydrogenase oxidizes OH, cleaves
  COOH -gt CO2
• also get NADH

Fig. 3
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TCA cycle reactions

• TCA cycle Reactions.
• B. a-ketoglutarate to Succinyl CoA
• Oxidative decarboxylation
• releases CO2
• Succinyl joins to CoA
• NADH formed
• GTP made from
• activated succinyl CoA

Fig. 3
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TCA cycle reactions

• TCA cycle Reactions.
• D. Oxidation of Succinate
• to oxaloacetate
• 2 e- from succinate
• to FAD-gt FAD(2H)
• Fumarate formed
• H2O added -gt malate
• 2 e- to NAD -gt NADH
• Oxaloacetate restored
• (common series of oxidations
• to CC, add H2O -gt -OH,
• oxidize -OH to CO)

Fig. 3
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III. Coenzymes are critical NAD

• Many dehydrogenases use NAD coenzyme
• NAD accepts 2 e- (hydride ion H-) -OH -gt CO
• NAD, and NADH are released from enzyme
• Can bind and inhibit different dehydrogenases
• NAD/NADH regulatory role (e-transport rate)

Fig. 5
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III. Coenzymes are critical for TCA cycle

• FAD can accept e- singly (as CC formation)
• FAD remains tightly bound to enzymes

Fig. 6 membrane bound succinate
dehydrogenase FAD transfers e- to Fe-S group and
to ETC
Fig. 4
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Coenzyme CoA in TCA cycle

• CoASH coenzyme forms thioester bond
• High energy bond
• (Fig. 8.12 structure of CoASH formed from
  pantothenate)

Fig. 7
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Coenzymes CoASH TPP

• Coenzymes CoASH, TPP
• (Figs. 8.11, 8.12)

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Coenzymes in a-ketoacid dehydrogenase complex.

• C. a-ketoacid dehydrogenase complex
• 3 member family (pyruvate dehydrogenase,
  branched-chain aa dehydrogenase)
• Ketoacid is decarboxylated
• CO2 released
• Keto group activated, attached CoA
• Huge enzyme complexes
• (3 enzymes E1, E2, E3)
• Different coenzymes in each

Fig. 8
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• a-ketoacid dehydrogenase enzyme complex
• 3 enzymes E1, E2, E3
• Coenzymes TPP(thiamine pyrophosphate).
• Lipoate, FAD

Fig. 9
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Lipoate is a coenzyme

• Lipoate coenzyme
• Made from carbohydrate, aa
• Not from vitamin precursor
• Attaches to NH2 of lysine of enzyme
• Transfers acyl fragment to CoASH
• Transfers e- from SH to FAD

Fig. 10
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Energetics of TCA cycle

• Energetics of TCA cycle overall net -DG0
• Some reactions positive
• Some loss of energy as heat (-13 kcal)
• Oxidation of NADH,
• FAD(2H) helps pull
• TCA cycle forward
• Very efficient cycle
• Yield 207 Kcal from
• 1 Acetyl -gt CO2
• (90 theoretical 228)
• Table 20.1

Fig. 11
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V. Regulation of TCA cycle

• Many points of regulation of TCA cycle
• PO4 state of ATP (ATPADP)
• Reduction state of NAD (ratio NADHNAD)
• NADH must enter ETC

Fig. 12
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Table 20.2 general regulatory mechanisms

• Table 20.2 general regulation metabolic paths
• Regulation matches function (tissue-specific
  differences)
• Often at rate-limiting step, slowest step
• Often first committed step of pathway, or
  branchpoint
• Regulatory enzymes often catalyze physiological
  irreversible reactions (differ in catabolic,
  biosynthetic paths)
• Often feedback regulation by end product
• Compartmentalization also helps control access to
  enzymes
• Hormonal regulation integrates responses among
  tissues
• Phosphorylation state of enyzmes
• Amount of enzyme
• Concentration of activator or inhibitor

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Citrate synthase simple regulation

• Citrate synthase simple regulation
• Concentration of oxaloacetate, the substrate
• Citrate is product inhibitor, competitive with S
• Malate -gt oxoaloacetate favors malate
• If NADH/NAD ratio decreases, more oxaloacetate
• If isocitrate dehydrogenase activated, less
  citrate

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Allosteric regulation of isocitrate Dehydrogenase

• Isocitrate dehydrogenase (ICDH)
• Rate-limiting step
• Allosteric activation by ADP
• Small inc ADP -gt large change rate
• Allosteric inhibition by NADH
• Reflect function of ETC

Fig. 13
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Other regulation of TCA

• Regulation of a-ketoglutarate dehydrogenase
• Product inhibited by NADH, succinyl CoA
• May be inhibited by GTP
• Like ICDH, responds to levels ADP, ETC activity
• Regulation of TCA cycle intermediates
• Ensures NADH made fast enough for ATP homeostasis
• Keeps concentration of intermediates appropriate

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VI. Precursors of Acetyl CoA

• VI. Many fuels feed directly into Acetyl CoA
• Will be completely oxidized to CO2

Fig. 14
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Pyruvate Dehydrogenase complex (PDC)

• Pyruvate Dehydrogenase complex (PDC)
• Critical step linking glycolysis to TCA
• Similar to aKGDH (Fig. 20.15)
• Huge complex
• Many copies each subunit
• (Beef heart 30 E1, 60 E2, 6 E3, X)

Fig. 15
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Regulation of PDC

• PDC regulated mostly by phosphorylation
• Both enzymes in complex
• PDC kinase add PO4 to ser on E1
• PDC phosphatase removes PO4
• PDC kinase
• inhibited by ADP, pyruvate
• Activated by Ac CoA, NADH

Fig. 16
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TCA cycle intermediates and anaplerotic paths

• TCA cycle intermediates - biosynthesis precursors
• Liver open cycle high efflux of intermediates
• Specific transporters inner mitochondrial
  membrane for pyruvate, citrate, a-KG, malate,
  ADP, ATP.

Fig. 17
GABA
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Anaplerotic reactions

• Anaplerotic reactions replenish 4-C needed to
  regenerate oxaloacetate and keep TCA cycling
• Pyruvate carboxylase
• Contains biotin
• Forms intermediate with CO2
• Requires ATP, Mg2 (Fig. 8.12)
• Found in many tissues

Fig. 18
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Amino acid degradation forms TCA cycle
intermediates

• Amino acid oxidation forms many TCA cycle
  intermediates
• Oxidation of
• even-chain fatty acids and
• ketone body not replenish

Fig. 19
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Key concepts

• TCA cycle accounts for about 2/3 of ATP generated
  from fuel oxidation
• Enyzmes are all located in mitochondrial
• Acetyl CoA is substrate for TCA cycle
• Generates CO2, NADH, FAD(2H), GTP
• e- from NADH, FAD(2H) to electron-transport
  chain.
• Enzymes need many cofactors
• Intermediates of TCA cycle are used for
  biosynthesis, replaced by anaplerotic (refilling)
  reactions
• TCA cycle enzymes are carefully regulated

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Nuclear-encoded proteins in mitochondria

• Nuclear-encoded proteins enter mitochondria via
  translocases
• Proteins made on free ribosomes, bound with
  chaperones
• N-terminal aa presequences
• TOM complex crosses outer
• TIM complex crosses inner
• Final processing
• Membrane proteins similar

Fig. 20
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Review question

• Succinyl dehydrogenase differs from other enzymes
  in the TCA cycle in that it is the only enzyme
  that displays which of the following
  characteristics?
• It is embedded in the inner mitochondrial
  membrane
• It is inhibited by NADH
• It contains bound FAD
• It contains fe-S centers
• It is regulated by a kinase