The Citric Acid Cycle, Krebs Cycle, Three Carboyxlic Acid Cycle

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The Citric Acid Cycle, Krebs Cycle, Three
Carboyxlic Acid Cycle

• Most mols enter the cycle as Acetyl-CoA
• There are three stages
• Acetyl-CoA production
• Acetyl-CoA oxidation
• Electron transfer
• Its distinguishing characteristics are
• The use of oxygen as the ultimate electron
  acceptor
• The complete oxidation of organic substrates to
  CO2 and H2O
• The conservation of much of the free energy as
  ATP
• The reactions of the citric acid cycle occur in
  the mitochondrial matrix, in contrast with
  glycolysis.
• An overview of the citric acid cycle
• Reactions of the citric acid cycle

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The Oxidative Decarboxylation of Pyruvate

• The condensation of Acetyl-CoA and oxaloacetate
  to form citrate
• Isomerization of citrate
• Oxidation of isocitrate
• Oxidation of ?-KG to succinyl-CoA
• Conversion of succinyl-CoA to succinate
• Oxidation of succinate to fumarate
• Hydration of fumarate to malate
• Oxidation of malate to oxalacetate

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The citric acid cycle

• Citric acid cycle (also called the Krebs cycle,
  TCA ) oxidizes Acetyl CoA to CO2 H2O
• Acetyl CoA
• Most mols enter the TCA cycle as Acetyl CoA. The
  cycle provides intermediates for biosynthesis..
  So, catabolism of proteins, fats and
  carbohydrates occurs in the 3 stages of cellular
  respiration.
• Stage I-----gt oxidation of f.a, Glc, some a.a
  yields Acetyl CoA
• Stage II-------gt oxidation of acetyl groups via
  the TCA cycle includes 4 steps in which electrons
  are abstracted.
• Stage III-------gt Electrons carried by NADH and
  FADH2 are funnelled into a chain of mitochondrial
  electron carriers-- respiratory chain- ultimately
  reducing O2 to H2O. This electron flow drives the
  synthesis of ATP, in the process of oxidative
  phosphorylation.

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TCA cycle
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Cycles distinguishing characteristics are

• The use of oxygen as the ultimate electron
  acceptor.
• The complete oxidation of organic substrates to
  CO2 and H2O.
• The conservation of much of the free energy as
  ATP.
• The reactions of the citric acid cycle occur
  inside mitochondria, in contrast with those of
  glycolysis, which take place in the cytosol.

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An overview of the citric acid cycle

• An overview of TCA cycle
• A 4C compound (oxaloacetate) condenses with a
  acetyl unit to yield a 6C tricarboxylic acid
  (citrate). An isomer of citrate is then
  oxidatively decarboxylated. The resulting 5C
  (a-ketoglutarate) is oxidatively decarboxylated
  to yield a 4C compound (succinate). Oxaloacetate
  is then regenerated from succinate.
• Reactions of the TCA cycle

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The oxidative decarboxylation of pyruvate.

• This is done by a multi-enzyme complex located in
  the mitochondrial matrix.
• Pyruvate dehydrogenase complex
• Pyruvate-----------------gt Acetyl CoA, a major
  fuel of the citric acid cycle, irreversible
  reaction.
• That means from Acetyl CoA we cannot make
  pyruvate that also explains why glucose can not
  be formed from Acetyl CoA in gluconeogenesis.

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PD complex

• 5 cofactors are involved in PD complex... All of
  which are coenzyme derived from vits..
• The regulation of this enzyme complex also shows
  how a combination of covalent modification and
  allosteric regulation results in precisely
  regulated flux through a metabolic step.
• Finally, the pyruvate dehydrogenase complex is
  the prototype for 2 other important enzyme
  complexes that well cover later.
• a-ketoglutarate dehydrogenase-------------gt TCA
  cycle
• a -ketoacid dehydrogenase--------gt a.a
  degradation

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Reactions of PD complex

• Step 1. Pyruvate reacts with the bound TPP of E1,
  undergoing deCO2 to form the Ohethyl derivative.
• Step2. The transfer of 2e- and the acetyl group
  from TPP to E2 to form acetyl thioester of the
  reduced lipoyl group.
• Step3. Transesterification -SH group of CoA
  replaces the SH group of E2 to yield AcetylCoA.
• Step4. E3 promotes transfer of 2H atoms from E2
  to the FAD of E3 restoring the oxidized form of
  the lypoyllysyl group of E2.
• Step5. The reduced FADH2 ON E3 TRANSFERS HYDRIDE
  ION TO NAD.

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Pyruvate Dehydrogenase
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OXIDATIVE DECARBOXYLATION OF PYRUVATE

• PDC is regulated by 2 mechanism.
• A. Product inhibition
• Inhibited by Acetyl CoA
• High concentrations of NADH
• B. Covalent modification PDC exists in 2 forms
• a) Active------gt nonphosphorylated
• b) Inactive------gt phosphorylated form.
  Phosphorylated and nonphosphorylated PDC can be
  interconverted by 2 separate enzymes.
• 1. A kinase
• 2. A phosphotase

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8 STEPS IN THE TCA CYCLE

• 1. The condensation of acetylCoA and oxaloacetate
  to form citrate
• The reaction uses an intermediate of the TCA
  cycle OA and produces another intermediate of the
  cycle (citrate). Thus, the entry of acetylCoA
  into the Krebs cycle does not lead to the net
  production or consumption of cycle intermediates.
• A refresher on enzyme nomenclature
• Synthases catalyze condensation reactions in
  which no ATP, GTP is required as an energy
  source.
• Synthetases also catalyze condensation reactions
  but this name implies that ATP or GTP is used for
  the synthetic reaction.
• Citrate syntase is inhibited by ATP, NADH,
  succinyl CoA derivatives of fatty acids.

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• Citrate, in addition to being an intermediate in
  the TCA cycle, has other functions
• 1. Provides a source of AcetylCoA for fa
  synthesis.
• 2. Citrate inhibits PFK, the rate limiting step
  in glycolysis, and activates Acetyl-CoA
  carboxylase the rate limiting enzyme for fa
  synthesis.

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2)ISOMERIZATION OF CITRATE

• Citrate is isomerized to isocitrate by a
  dehydration step followed by a hydration step.
  Cis-aconitate occurs as an enzyme-bound
  intermediate.

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3.OXIDATION OF ISOCITRATE

• Isocitrate dehydrogenase catalyzes the
  irreversible oxidadite deCO2 of isocitrate
  yielding the first of 3 NADH mols produced by the
  cycle and CO2.
• - Enzyme activated by ADP. Elevated levels of
  mitochondrial ADP signals a need for the
  generation of more high-energy phosphate (ATP).
• - The enzyme is inhibited by ATP and NADH, which
  are increased when the cell has abundant energy.

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4. OXIDATION OF a-KG TO SUCCINYLCOA

• irreversible reaction.
• Enzyme a -KGDC, it is similar to PDC reaction.
• It also has 3 enzymes (analogous to E1. E2, E3)
  and 5 cofactors. (TPP, lipoic acid, FAD, NAD, and
  CoA)
• Enzyme is inhibited by ATP, GTP, NADH and
  succinylCoA, but is not regulated by
  phosphorylation/dephosphorylation reactions as
  described for PDC, 2nd CO2 and 2nd NADH are
  produced.

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5. CONVERSION OF SUCCINYL COA TO SUCCINATE

• This reaction is coupled to the phosphorylation
  of GDP to GTP. The energy content of GTP is the
  same as that of ATP, because 2 nucleotides are
  interconvertible by the nucleoside diphosphate
  kinase reaction.
• This is an example of substrate -level
  phosphorylation in which the ATP production is
  coupled to the conversion of substrate to
  product, rather than resulting from
  respiratory-chain.

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6.OXIDATION OF SUCCINATE TO FUMARATE

• FAD rather than NAD is the e-acceptor, since the
  reducing power of succinate is not sufficient to
  reduce NAD. Malonate, a dicarboxylic acid that
  is a structural analog of succinate,
  competitively inhibits succinate dehydrogenase.

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7.HYDRATION OF FUMARATE TO MALATE
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8.OXIDATION OF MALATE TO OXALOACETATE
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TCA cycle
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STOICHIOMETRY OF THE CYCLE

• Summary of the reactions
• 1. Two carbon atoms enter the cycle as acetyl CoA
  and leave as CO2.
• 2. The TCA cycle does not involve the net
  consumption or production of OA or any other
  intermediate of the cycle.
• 3. Four pairs of e- are transferred during one
  one turn of the cycle 3 pairs of e- reducing
  NAD to NADH and one pair reducing FAD to FADH2.
• ATP FORMATION IN THE AEROBIC OXIDATION OF A
  MOLECULE OF GLC VIA GLYCOLYSIS, THE PDC REACTION
  AND THE TCA CYCLE

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CITRIC ACID CYCLE COMPONENTS ARE IMPORTANT
BIOSYNTHETIC INTERMEDIATES.

• The TCA cycle is an amphibolic pathway, meaning
  it serves in both catabolic and anabolic
  processes. It also provides precursors for many
  biosynthetic pathways. But if this is the case ,
  we have to replace the ones used for the
  biosynthesis of some molecules. Those reactions
  which replenish TCA acid cycle intermediates are
  called anaplerotic reactions. Under normal
  circumstances there is a dynamic balance between
  the reactions by which the cycle intermediates
  are used and those by which they are replenished
  by the anaplerotic reactions. So that the
  concentrations of the citric acid cycle
  intermediates remain almost constant.
• Given the number of biosynthetic products
  synthesized from the TCA cycle intermediates,
  this cycle serves a critical role apart from its
  role in energy yielding metabolism.

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ATP Formation in the Aerobic Oxidation of GLC
Via Glycolysis, the PDC Reaction and the TCA
Cycle
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ANAPLEROTIC REACTIONS

• REACTION TISSUE, ORGANS
• 1)
• 2)
• 3)
• 4)
• They are all reversible. When TCA needs OA,
  pyruvate is carboxylated to OA. Free energy is
  required to attach CO2 to pyruvate comes from
  ATP. Carboxylation of pyruvate also requires,
  like in other carboxylation reactions BIOTIN,
  which is a prosthetic group of pyruvate
  carboxylase.

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THREE ENZYMES OF THE TCA CYCLE ARE REGULATED

• The TCA cycle is under tight regulation. 3
  factors are important for the rate of flux
  through the cycle.
• 1. Substrate availability
• 2. Product inhibition
• 3. Allosteric feedback inhibition of early
  enzymes by later intermediates in the cycle.

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There are 3 irreversible steps in the cycle,
therefore potential sites for control. Those are
catalyzed by

• Citrate synthase
• Isocitrate dehydrogenase
• a-KG dehydrogenase.
• Each can become a rate limiting step under
  certain circumstances. When acetyl CoA and OA are
  availabele or not , citrate formation increase or
  decrease.
• NADH increases (a product of the oxidation of
  isocitrate and a -KG) , NADH/NAD increases,
  those dehydrogenase reactions are severely
  inhibited.

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Regulation of TCA cycle
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The TCA cycle is a source of biosynthetic
precursors
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Regulation of CarbohydrateMetabolism
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The disruption of pyruvate metabolism is the
cause of beriberi and heavy metal poisoning

• TPP deficiency causes beriberi
• Hg, Ar, and Pb have high affinity for -SH
• Lipoic acid is one of the cofactors in PDC
• PDC becomes inactive when lipoic acid is bound to
  heavy metals.
• CNS solely depends on Glc metabolism therefore
  effected by heavy metal poisoning.

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The glyoxylate cycle permits AcetylCoA to be
incorparated into carbohydrates

• The glyoxylate cycle , a modification of the TCA
  cycle, is a biosnthetic pathway that leads to the
  formation of glucose from AcetylCoA.
• It occurs in
• Plants
• Bacteria
• Yeast
• Not in Animals

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glyoxylate cycle

• In oily seed plants, the glyoxylate cycle is
  especially active. The glyoxylate cycle can be
  regarded as a shunt within the TCA cycle.
• 1. The 6C intermediate isocitrate, rather than
  undergoing decarboxylation, is converted to the
  4C mol succinate and 2C mol glyoxylate in a
  reaction catalyzed by isocitrate lyase, the first
  of the 2 enzymes in this cycle.

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Glyoxylate cycle
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Glyoxylate cycle
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More about glyoxylate cycle

• In plants the enzymes of the glyoxylate cycle are
  in the membrane bound organelles, called
  glyoxysomes. Glyoxylate enzymes are not present
  in animal cells, thus animals can not sustain
  growth on acetylCoA or 2C mols, such as acetate.
  
• Role of the glyoxylate cycle
• 4C and 6C compounds are made from 2C compounds
• Glucose is made from acetate
• It is also essential reaction sequence for
  seedlings of fat storing in plants.
• TCA and Glyoxylate cycles are coordinately
  regulated.