Topic 12 - TCA Cycle Flashcards
01 Citrate Synthase
Acetyl-CoA + OAA –> **Citrate + CoA-SH (ΔG = -31.4 kJ/mol**, must be regulated.)
Lyase.
- *+ Effectors**: AMP/ADP.
- *- Effectors**: ATP, NADH, Citrate, Succinyl-CoA (has a similar structure, binds to pocket).
Mechanism: Aldol condensation. Extends the 4-Carbon OAA to a 6-Carbon molecule. Rate-limiting step of the cycle. Occurs via deprotonation of the acetyl group and nuc attack by the resulting carbanion on the C=O of OAA. Reaction is highly spontaneous due to thiol ester hydrolysis. Special, induced fit mechanism leads to specific order of substrate binding to prevent futile hydrolysis (same as HK in glycolysis).
02 Aconitase
Citrate –> Isocitrate (ΔG = +6.7 kJ/mol)
Isomerase.
Mechanism: Converts the tertiary alcohol to a secondary alcohol. The two following reactions (both highly spontaneous) drive it forward by consuming isocitrate. This reaction prepares the substrate for oxidative decarboxylation in the next reaction. A 2o alcohol is more readily oxidized to a C=O than a 3o alcohol, and the resulting C=O would be beta to the COO- group to be removed. This faciliates decarboxylation. Stereospecific - a 3-point binding of the substrate to the enzyme orients it in a way that ensures that the OH group always moves to the end of the molecule that originated from OAA, never to the end that originated as the acetyl group.
03 Isocitrate Dehydrogenase (IDH)
Isocitrate + NAD+ –> α-KetoGlutarate (α-KG) + NADH + H+ + CO2 (ΔG = -20.9 kJ/mol, must be regulated.)
Oxidoreductase #1.
Oxidation & Decarboxylation. Produces NADH.
+ Effectors: AMP/ADP, NAD+, Ca2+ (stimulates glycogen breakdown, making glucose available for metabolism. Also triggers muscle contraction, which requires ATP hydrolysis).
- Effectors: ATP, NADH (too much –> take NADH and bind to isocitrate dehydrogenase to shut down reaction). In E. coli, reversible phosphorylation inhibits binding of isocitrate and prevents its catalysis.
Mechanism: Highly spontaneous oxidative decarboxylation reaction yields the first energy currency of the cycle (NADH) and results in oxidation of another carbon to CO2. Generates a 5-Carbon molecule.
04 α-KetoGlutarate Dehydrogenase
04 α-KetoGlutarate (α-KG) + NAD+ –> Succinyl-CoA + NADH + H+ + CO2 (ΔG = -33.5 kJ/mol, must be regulated)
Oxidoreductase #2.
Produces NADH = 2.5 ATP.
+ Effectors: Ca2+ (stimulates glycogen breakdown, making glucose available for metabolism. Also triggers muscle contraction, which requires ATP hydrolysis).
- Effectors: ATP, NADH, Succinyl-CoA.
Mechanism: Regenerates the 4-Carbon molecule (CoA excluded, although the Carbons that are oxidized come from OAA, not CoA!). Results in succinyl-CoA, a high energy thieoster! Similar to PDH complex in which it consists of 3 types of enzymes aggregated together. The only difference is that E1 must accommodate the larger 5-Carbon α-keto-diCarboxylic acid and E2 must accommodate the larger acyl group, succinate. E3 catalyzes the exact same reaction as in PDH.
- *E1** = TPP-connected, stabilizes intermediate. Loss of CO2 –> makes it a carbanion, which is stabilized by E1.
- *E2** = Lipoamide - transfer carbanion.
- *E3** = Helps reoxidize enzyme (exact same enzyme as in PDH complex).
- *Regulation**: Regulation is analogous to PDH.
1. Succinyl-CoA regulates TCA cycle… how?
05 Succinyl-CoA Synthetase
Succinyl-CoA + GDP + Pi –> Succinate + GTP (ΔG = -2.9 kJ/mol)
Ligase: New bond at expense of ATP/GTP.
Produces GTP via substrate-level phosphorylation.
Mechanism: Hydrolysis of the thiol ester drives GTP synthesis from GDP + Pi (condensation reaction). (Interconversion of ATP & GTP results in ΔG ~ 0).
06 Succinate Dehydrogenase (SDH)
Succinyl-CoA + FAD –> Fumarate + FADH2 (ΔG = +0.4 kJ/mol)
Oxidoreductase #3. Prosthetic group: FAD.
Produces FADH2 = 1.5 ATP.
Mechanism: Oxidation of succinate (an alkane) to fumarate (an alkene). The oxidation of an alkane to an alkene utilizes FAD instead of NAD as the electron acceptor b/c electrons must be passed from a compound of lower reduction potential to one of higher reduction potential. Fumarate/succinate has a higher reduction potential than NAD+ but lower than FAD, so fumarate/succinate must pass its electrons to FAD.
The microenvironment provided by SDH gives the covalently-bound FAD a higher reduction potential than free FAD/FADH. If NAD+ were the electron acceptor, the reaction would be highly unfavorable. (ΔE = Eelectron acceptor - Eelectron donor, ΔE = -ΔG/nF where ΔG is negative for spontaneous oxidation-reduction reactions). SDH is the only membrane-bound enzyme of the TCA cycle. It is also part of Complex II in ETC. All other enzymes in the TCA cycle are soluble in the matrix.
07 Fumarase
Fumarate –> L-Malate (ΔG = -3.8 kJ/mol)
Hydrolase.
08 Malate Dehydrogenase (MDH)
L-Malate + NAD+ –> Oxaloacetate (OAA) + NADH + H+ (ΔG = +29.7 kJ/mol).
Oxidoreductase #4. Produces NADH (2.5 ATP).
Mechanism: The energy of the Citrate Synthase reaction (the first reaction of the TCA cycle) is necessary to pull this reaction forward. ΔG = +29.7 kJ/mol (MDH) + -31.4 kJ/mol (Citrate Synthase) = -1.7 kJ/mol. It is reversible.
What is the net effect of the TCA Cycle?
Total (1 cycle): 2 CO2 + 1 ATP(1) + 1 FADH2(1.5) + 3 NADH(2.5) = 10 ATP
Total (2 cycles): 4 CO2 + 2 ATP(1) + 2 FADH2(1.5) + 6 NADH(2.5) = 20 ATP
What are the regulatory steps of the TCA Cycle?
- Citrate Synthase
- Isocitrate Dehydrogenase
- α-KetoGlutarate Dehydrogenase
What are the names of the molecules in the TCA Cycle?
- *Can**: Citrate
- *Adam**: *cis-Aconitate
- *Intrigue**: Isocitrate
- *A**: alpha-ketoglutarate
- *Super**: Succinyl-CoA
- *Sexy**: Succinate
- *Foxy**: Fumarate
- *Mama**: Malate
- *Ok!**: Oxaloacetate
What are the names of the enzymes in the TCA Cycle?
- So: Citrate Synthetase
-
At: Aconitase
* *Another**: Aconitase - Dance: Isocitrate Dehydrogenase
- Devon: α-ketoglutarate Dehydrogenase
- Sipped: Succinyl-CoA Synthetase
- Down: Succinate Dehydrogenase
- Five: Fumarase
- Drinks: Malate Dehydrogenase
The key to remembering how many CO2 or NADH is made: 3 out of the 4 dehydrogenase enzymes will remove 2 H atoms(and an electron) from the molecule an put it onto a NAD, and remove a CO2 molecule. The only exception to this rule is the enzyme that produces Fumarate, Succinate Dehydrogenase. It actually places 2 H atoms on an FAD molecule instead of an NAD.
What does the “amphibolic nature” of the TCA Cycle refer to? How are its intermediates utilized as precursors for biosynthesis?
Even though the TCA Cycle itself does not result in the net synthesis of any carbon compounds, some of its intermediates are utilized as precursors for biosynthesis:
- Acetyl-CoA –> Fatty acids, Cholesterol. Cholesterol –> Steroids.
- α-KG –> Glutamate –> Glutamine, Proline, Arginine. Glutamine –> Purine nucleotides.
- Succinyl-CoA –> Porphyrins.
- OAA –> PEP –> Glucose. (Gluconeogenesis)
- OAA –> Aspartate –> Lysine, Threonine, Methionine, Isoleucine, Asparagine. Aspartate –> Pyrimidine nucleotides.
How are TCA intermediates replenished?
Various degradative pathways generate TCA Cycle intermediates:
- Oxidation of odd-chain fatty acids –> Succinyl-CoA
- Degradation of Ile, Met, Val –> Succinyl-CoA
- Degradation of other aa –> Fumarate
- Amino-transfer reactions –> OAA & α-KG
What are “anaplerotic” reactions?
They ensure the ability to achieve a net production of OAA for the TCA Cycle even if degradative pathways are not currently occurring.
-
Pyruvate Carboxylase: Pyruvate + HCO3-/CO2 + ATP –> OAA + ADP + Pi
-Uses biotin (Vitamin H)
+Effector: Acetyl-CoA -
Glyoxylate Bypass:
a. Isocitrate Lyase: Isocitrate –> Succinate + Glyoxylate
b. Malate Synthase: Glyoxylate + Acetyl-SCoA + H2O –> Malate + CoA-SH + H+
