TCA Cycle, Tracking Carbons, Anaplerotic Reactoin and Regulations

Summary of the energetics of the TCA

• 4 oxidation steps occur

  • 3NAD+ to NADH

  • 1 FAD to FADH2

  • 1 substrate-level phosphorylation by succinyl-CoA synthetase creates GTP

  • Oxalacetate is regenerated

• From glucose

  • Glycolysis: 2 ATP and 2 NADH

  • PDH: 2NADH

  • 2 Citric acid cycle turns creates

    • 6NADH, 2 FADH2 and 2 GTP

Carbon Tracking

• Follows the fate of individual carbon atom as they move through metabolic reaction

• Done using isotopic labels on specific carbons on the substrate

• Reveals which carbons are lost as CO2 and what remain in intermediates in each TCA cycle turn

Essential to understand

• Metabolic flux

  • How fast metabolic pathways run in the cell

• Carbon balance

  • Tracks where every carbon atom goes as molecules are transformed during emtabolism

  • Ensure the number and fate of carbon entering and leaving pathway is accounted for

• Labeling studies

  • Insight into the sequence of intermediates in metabolic pathway

Fate of Carbons from Glucose to pyruvate and acetyl CoA

• Carbon 1 and 6 had the phosphate tag

• DHAP: C1-C3, GAP C4-C6

• Carboxylate group on GAP is either C3 or C4

• Carbonyl group on GAP is C2 or C5

• Methyl group on GAP is C1 or C6

Pyruvate to acetyl-CoA

• Carboxylate leaves as carbon dioxide, C3 and C4 leave

• C2 or C5 has thioester group

• C1 or C6 is the methyl group

Tracking C2/5 from glucose through the TCA Cycle

GlC 2/5 are lost as CO2 by combined IDH and alpha-KGDH on the second cycle, 100% lost on the second cycle but each individual carbon has a 50/50 change to be lost in either step

Cycle 1

• C2/C5 is a carboxylate farthest from Co-A at the production of succinyl-CoA step

• Fumarate is symmetric so the hydration by fumarase can occur at either C2 or C3

• The labelled carbon is split 50/50 between the two carboxylate groups in malate since hydration can occur at C2 or C3 the labelled carbon is has a split probability

Cycle 2

• The 2 carboxylate carbons from OAA are labelled 50/50

• Both are lost as CO2 in the IDH and alpha-KGDH step of cycle 2

Tracking C1/C6 from Glucose through the TCA cycle

• Glc1/6, lost by combined IDH and alpha KGDH step on the subsequent steps

  • 50% of label is lost on 3rd cycle, 25% each step

  • 25% of label is lost on 4th cycle, 12.5% each step

  • 12.5% of label is lost on 5th cycle, 6.25% each step

  • 6.25% of label is lost on 6th cycle, 3.13% each step

• Pattern continues infinitely, each cycle release 50% of remaining label as CO2

Cycle 1

• From glucose/pyruvate/acetyl CoA, C1 and C6 is the methyl group on acetyl CoA which directly connects to the carbonyl carbon in OAA to form 6C citrate

• Since fumarate is symmetric C1/C6 can be either of the alkene carbons, when hydration occurs there's a 50/50 probability than it hydrated the label carbon or it's neighbour

• No label is lost

• On oxalacetate the carbon is either the methylene group or carbonyl

Cycle 2

• On citrate the carbon is either the carboxylate or the methylene

• On fumarate it is either the carboxylate or CH group

• When fumarase hydrates the label is split 25% to all carbons on malate

• No label lost as CO2

Cycle 3

• IDH/alpha-KGDH release 50% of the carbon label, the remaining carbons from citrate remain on the carboxylate and CH carbon in fumarate

• At fumarate the label is split 12.5% to each of the four carbons of malate

Cycle 4

• IDH/alpha KGDH steps release a combined 25% of carbon label

• Remaining carbon from citrate are retained in the carboxylate and CH group in fumarate

• Fumarase, the label is split 6.25% to each of four carbons of malate

Pyruvate carboxylation entry

• When pyruvate is converted directly to oxalacetate by pyruvate carboxylase, the new carbon from CO2 is incorporated into OAA and then enters the TCA cycle

  • Serves as the starting point for all TCA cycle intermediates

 Anaphoretic Reactions

• Replenishes the supply of TCA intermediates, specifically the 4C dicarboxylate pool as it is continuously used for biosynthesis

• Anaplerosis

  • Anaplerotic reactions replenish the supply of TCA intermediates

• Anaplerotic reactions

  • Used for replenishing 4C dicarboxylate pool

    • When 4C dicarboxylates are used for biosynthesis, pyruvate carboxylase makes OAA to keep the TCA cyle running

4 reactions

• Pyruvate carboxylase

  • Pyruvate + HCO3- + ATP > oxaloacetate + ADP + Pi

  • In the liver and kidney

• PEP carboxykinase

  • PEP + CO2 + GDP > oxaloacetate + GTP

  • In the heart and skeletal muscle

• PEP carboxylase

  • PEP + HCO3- > oxaloacetate +Pi

  • In higher plants, yeast and bacteria

• Malic Enzyme

  • Pyruvate + HCO3- + NAD(PH) > malate + NAD(P)+

  • Widely distributed in eukaryotes and bacteria

Pyruvate Carboxylase Reaction

• anapleurotic ping-pong reaction

  • Each reaction, substrate bind and products are release in a back and forth matter, each reaction modifies the enzyme for the subsequent reaction

  • Pyruvate + HCO3- + ATP > oxaloacetate + ADP + Pi

• PC catalyzes carbon-fixing reaction that has a anapleurotic function to bring new carbons into the TCA cycle

• In gluconeogenesis: CO2 is a temporary tag, is added to pyruvate by PC to form OAA then release again by PEP carboxin's in the next step

• PC can serve an anaplerotic role, OAA produced by PC can enter the TCA and serve as the starting point for all TCA cycle intermediates and their derived metabolites

PC first half reaction At E1 site

HCO3- binds to the enzyme, ATP cleavage drives attachment of CO2 to enzyme

• A bicarbonate kinase forms carboxyphosphate (high energy intermediate)

• PC transfers the CO2 group to form carboxybiotin, a high energy CO2 carrier ebfore adding it to pyruvate

  • Biotin cofactor has an amino group to hold CO2

  • Biotin has a tether attached to the enzyme, enables it to travel between active site for the 2 partial reactions at site 1 and 2

PC second half reaction at E2 site

• Deprotonated of the pyruvate alpha Carbon makes an enolate, it serves a nucleophile to form a new bond

• The carbon in Biotin-CO2 is an electrophilic center and the enolate carbon is a nucleophile

• A new C-C bond is created between the C in CO2 and alpha carbon in enolate to create oxaloacetate

Track Carbon atoms from pyruvate to OAA

• The carboxylate is added onto C1/C6 of pyruvate

  • PC route

• The acetyl-COA route, C1/C6 forms a C-C bond by enolate addition to C2/C5 of OAA

Glutamate

• Amino acid providing anaplerosis

• Amino acid from proteins in diet can be a carbon source when the alpha-amino group is removed

• The amino group is removed and glutamate dehydrogenase oxidizes the alpha carbon to create a keto to make, alpha-ketoglutarate

  • Uses NAD+ to NADH

Glu DH: Oxidative deamination

• Dehydrogenation of amine to imine on glutamate

  • NH3+-C-H to NH2=C

  • a proton from NH3 and proton from the carbon leaves by NAD+

• Imine hydrolysis

  • 2 C=N bonds is replaced by two C=O bonds

  • Water is used as substrate, NH3 leaves

  • Water attacks the imine and NH3 departs to leave a keto

• formate keto-glutarate

Alpha-KG collects the alpha-amino groups from other amino acids

• Aminotransferase family of enzymes

• Each enzyme combines a specific amino acid with alpha KG

• Amino acid undergoes oxidative deamination and alpha KG collects the amino group to make glutamate

• Reaction is cataplerotic, opposite fo anaplerotic as it removes TCA intermediates

• Alpha KG oxidizes other amino acids

Alanine amino-transferase

Balance alpha KG removal with potential for indirect OAA generation

• by producing more alpha KG from Glutamate it can take the amino group from alanine to produce more pyruvate which can increase OAA generation

Alanine amino-transferase

Alanine amino-transferase: Balance alpha KG removal with potential for indirect OAA generation

• Anapleotic reaction

  • Glutamate (Glu DH) + NAD+ + H2O >  Alpha-KG + NADH + NH3

  • 5 carbons

• Cataplectic  reaction

  • alphaKG + 3C Alanine (Ala amino-transferase) > Glutamate + pyruvate

  • Alanine amino-transferase

    • Doesn't change any C-C bonds

    • Balances cataplerotic alpha-KG removal with pyruvate production to feed back into TCA through anapleotic PC activity

  • Pyruvate then becomes OAA by pyruvate carboxylase

TCA cycle Regulation

• Regulation targets the irreversible exergonic steps of catabolism, including the PDH complex and the 3 TCA cycle enzyme that make/break C-C bonds

  • Citrate synthase

  • Isocitrate dehydrogenase

  • Alpha-KG dehydrogenase

Energy indicators

• High ATP/NADH inhibit the enzymes, signals ample energy

• ADP/AMP and Ca2+ activate them, specifically in muscle contraction

Production and substrate control

• Build up of citrate, succinyl-CoA, acetyl CoA and NADH inhibits upstream enzymes

• Adequate OAA and Acetyl-CoA are required to sustain flux

Negative Regulators:

energy capture molecules are inhibitors of catabolism

• PDH, CS, IDH and alpha-KGDH are targeted

• Energy store abundance suppresses aerobic catabolism

• Negative regulators = feedback inhibitors = energy store products

  • NADH, Acetyl-CoA and Succinyl-CoA

    •  molecules store energy that was captured during oxidative decarboxylation dehydrogenase reactions

  • ATP

    • Indicate success in catabolism

  • Citrate and fatty acids

    • Reflects success in enetry to TCA

• ATP and citrate are also feedback inhibition of committed steps in glycolysis (PFK-1)

Positive Regulators, Energy shortage

• Feed-forward activators = precurosrs of energy stores

• NAD+ and free CoA

  • Reactans of oxidative decarboxylation dehydrogenase

• ADP and AMP

  • Waste products of ATP usage that need to be re-charged into ATP

• Activated by signals of energy demand, ADP/AMP and Ca2_, ensures the cycle accelerates only when ATP is needed

Glyoxylate cycle: How plants turn fat into sugar

• Net synthesis of carbohydrate (glucose) from acetyl-CoA (fat_

• It bypasses the 2 decarboxylation steps of TCA cycle (isocitrate > alpha-KG > succinyl-CoA) to prevent loss of carbon as CO2

• Acetyl co-A enters, Isocitrate lyase splits isocitrate into succinate 4C and glyoxylate 2C

  • Malate synthase condenses glyoxylate with another acetyl-CoA to form malate

• Succinate is enter the TCA cycle to convert into OAA to be used in gluconeogenesis

  • The glyoxylate cycle preserves acetyl-CoA carbons allowing Glucose synthesis from fat

Specialize Organelles: Glyoxysomes

• The enzymes of the glyoxylate cycle exists as 2 isozymes, one is localized to the mitochondria and the other is in the glyoxysomes

• It is a organelles that isn't present in all plant tissues at all times, form in lipid-rich seeds during gemination when the seedling cannot produce glucose by photosynthesis

• Glyoxysomes house a complete set of enzymes needed to break down fatty acids stores in seed oils

  • Link fat metabolism directly to the production of glucose precursors