PS2_14: The PDC and the CAC

The Pyruvate Dehydrogenase Complex (PDC) and the Citric Acid Cycle (CAC)

Includes Amino Acid Metabolism

Citric Acid Cycle Overview

Central hub of metabolism.
Other names: TCA Cycle, Krebs Cycle (named after Hans Krebs).
Series of chemical reactions extracting electrons (carrying energy) from Acetyl-Co-A (2-Carbon molecule).
Metabolism of lipids, amino acids, and carbohydrates converges at the citric acid cycle.
The citric acid cycle also supplies key metabolic intermediates, interfacing with other metabolic pathways, continuously replenishing the cycle.

Learning Objectives

Understand the biological, biochemical, and chemical rationale of the citric acid cycle.
Understand how pyruvate and amino acids can be converted into substrates of the CAC.
Understand how flux through the CAC is regulated.
Understand the chemical mechanisms associated with enzymatic transformations during the CAC.
Understand the anapleurotic reactions.

Terminology

Dehydrogenases: Enzymes catalyzing the loss of a hydrogen atom from the donor.
Oxidase: Enzymes using molecular oxygen as the electron acceptor.
Oxygenase: Enzymes catalyzing a direct combination of oxygen with the substrate.

Location

In eukaryotes, all steps occur in the mitochondria.
Most enzymes of the citric acid cycle are in the matrix.
One critical exception exists.

Cellular Respiration vs. Fermentation

Anaerobic organisms perform fermentation, converting pyruvate into ethanol or lactic acid, which is less efficient.
Complete combustion of glucose to $CO2 + H2O$ is exergonic (~2900 kJ/mol).
Combustion of ethanol (2 molecules per glucose) releases ~1330 kJ/mol of energy.
~90% of the energy in the original glucose molecule remains in the products of fermentation.
Organisms using oxygen convert the 3C product of glycolysis to a 2C thioester (acetyl-coA).
Energy released in going from 3C → 2C is captured in the form of reduced NADH.
The resulting 2C molecule is fully oxidized to $CO_2$ , releasing available free energy.
This energy is used to make ATP, powering critical cell processes.

Stages of Cellular Respiration

Stage 1: Oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA.
Stage 2: Oxidation of acetyl groups in the citric acid cycle; four steps involve electron abstraction. No oxygen is directly consumed.
Stage 3: Electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or plasma membrane-bound in bacteria) electron carriers—the respiratory chain—ultimately reducing $O2$ to $H2O$ . This electron flow drives ATP production.

Pyruvate Dehydrogenase Complex

Pyruvate is not a substrate for the citric acid cycle and must first be converted to acetyl-coA by the Pyruvate Dehydrogenase Complex.
Aerobic organisms use glycolysis as the first step in carbohydrate metabolism and usually do not ferment unless tissues are deficient in oxygen.
Aerobic organisms extract more energy from glucose because:
- They can use the energy from NADH (instead of using it for fermentation).
- They can extract energy from pyruvate, which is not possible in anaerobic metabolism.
Four steps for carbohydrate metabolism:
- Pyruvate Dehydrogenase: Converts Pyruvate (3 carbons) to Acetyl-coA (2 Carbons).
- Citric Acid Cycle: Converts Acetyl-coA (2 Carbons) to carbon dioxide (1 Carbon).
- Electron Transport Chain: Uses energy to create a proton gradient.
- Oxidative Phosphorylation: Uses proton gradient to power ATP synthesis.

Pyruvate Dehydrogenase Complex (PDH Details)

Enzymatic machinery for converting pyruvate to acetyl-coA.
Pyruvate is generated in the cytoplasm, but PDH reactions happen within the mitochondrial matrix.
The mitochondrial pyruvate carrier spans the inner mitochondrial membrane, allowing free diffusion of pyruvate into the matrix.
The mitochondrial pyruvate carrier is coded for by 2 genes: MPC1 and MPC2.
In certain cancers (gliomas), the MPC genes are heavily mutated, potentially contributing to the Warburg effect.

Catalysts in the Citric Acid Cycle

Enzymes: Speed up reaction rates.
Small molecules (Krebs' proposal): Regenerated in a cyclic process, acting as a "mold" or "scaffold" for molecules to be assembled or disassembled.
The catalyst is destroyed but then regenerated.

Pyruvate Oxidation States

Two molecules of pyruvate are generated from each molecule of glucose.
Pyruvate has two faces: one energy-depleted (almost like $CO_2$ ) and one energy-rich.
Two molecules of NADH were produced per glucose during glycolysis.
The term "aerobic metabolism" can be misleading because many steps are not oxygen-requiring but converge on an oxygen-dependent process.

Pyruvate to Acetate

The bond to break is between a carbon and the carbonyl group.
To overcome this, a "pretend" carbonyl group is installed as a temporary electron sink.

Thiamine Pyrophosphate (Ylid)

Critical cofactor for the pyruvate dehydrogenase complex.
Analogous reactions seen in ethanol fermentation and the Pentose Phosphate Pathway.

Pyruvate Decarboxylase

Chemical mechanism of the decarboxylation of pyruvate and the role of the TPP (ylide) cofactor.
The ylide forms a transient interaction with the pyruvate, allowing electrons to temporarily reside on it, enabling the reaction to go to completion (protonating the substrate to form acetaldehyde).

Thioesters

Acetyl-CoA is a thioester with a large, negative, standard free energy of hydrolysis.
Esters (oxygen esters) have limited resonance.
The sulfur atom is larger; there is even less C-S orbital overlap, leading to no effective resonance taking place. As a result, the bond is weaker and breakage is more spontaneous.

Lipoic Acid

Another important cofactor is lipoic acid (or lipoamide, when attached to a lysine residue in a protein).
The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the PDH complex).
The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group.
Biotin was the first example of a cofactor with a long spacer arm.

Pyruvate Dehydrogenase Overall Reaction

The product is a thioester with a high free energy of hydrolysis due to the block on resonance from the thioester.

Pyruvate Dehydrogenase Complex Structure

Three-dimensional image of PDH complex, showing the subunit structure: E1 (pyruvate dehydrogenase), E2 (dihydrolipoyl transacetylase), and E3 (dihydrolipoyl dehydrogenase).
The core consists of 60 molecules of E2, arranged in 20 trimers to form a pentagonal dodecahedron.
The lipoyl domain of E2 reaches outward to touch the active sites of E1 molecules arranged on the E2 core.
Several E3 subunits are also bound to the core, where the swinging arm on E2 can reach their active sites.
Coenzymes: CoA, NAD+, TPP, Lipoate, FAD

Oxidative Decarboxylation by PDH Complex

Step 1: Pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E1), undergoing decarboxylation to the hydroxyethyl derivative.
Step 2: Pyruvate dehydrogenase also carries out the transfer of two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced lipoyl group.
Step 3: Transesterification in which the -SH group of CoA replaces the —SH group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group.
Step 4: Dihydrolipoyl dehydrogenase (E3) promotes the transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2.
Step 5: The reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH. The enzyme complex is now ready for another catalytic cycle.

TPP / E1

The carbanion of TPP installs itself into the pyruvate via a nucleophilic reaction.
Purpose: The thiamine pyrophosphate group stabilizes the carbanion intermediate in the decarboxylation of pyruvate.

Lipoamide / E2

The acetate group (and its electrons) are transferred to the oxidized lipoamide to form the reduced acetlylipoamide.
This is made possible by the long swinging arm of the lipoamide, which can carry the substrate between active sites.

Acetyl-CoA / E2

The acetate electrophile is attacked by the coenzyme-A nucleophile.
The electrons are left behind on the lipoamide.
Acetyl-coA, which can enter into the citric acid cycle, is created.

FADH2 NADH/ E3

FAD recovers the electrons, and their energy, regenerating the oxidized lipoamide.
FAD is covalently attached to the protons.
Electrons moved to a soluble carrier, NAD+.
Generally, the potentials of NAD+/NADH is constant and unaffected by environment.
The flavins are bound much more tightly to the protein.
The actual reduction potential of the flavins is dependent upon how tightly they interact with their proteins and the chemical environment surrounding the flavin ring.
At the end of the Pyruvate Dehydrogenase Complex:
- (1) A substrate for the Citric Acid Cycle has been formed.
- (2) The electrons, and their associated energy, have been captured from the conversion of pyruvate to acetyl-coA.

Regulation of Pyruvate Dehydrogenase Complex

Inhibited when ATP/ADP is high and when NADH/NAD+ is high.

Regulation of the PDH Complex - I

Two distinct modes of regulation: allosteric and phosphorylation.
Proteins have their activity modulated both by signaling (phosphorylation) and allosteric effectors.
All organisms use allosteric regulation regulation by phosphorylation is more specific and is only used by mammals.
Pyruvate, NAD+, and coA activate, while NADH and Acetyl-CoA inhibit.

Regulation of the PDH Complex -II

E1 of the PDH is regulated by phosphorylation; when it is phosphorylated, activity is off.
PDH kinase phosphorylates PDH E1, turning it off.
PDH Phosphatase dephosphorylates PDH E1, restoring activity.
NADH, acetyl-CoA, and ATP inhibit PDH Kinase whereas NAD+, CoA, ADP, and $Ca^{2+}$ activate PDH Kinase.
$Ca^{2+}$ , $Mg^{2+}$ , and insulin (adipocytes) activate PDH Phosphatase.

The Mg Sensor

Magnesium stimulates the PDH Phosphatase, activating the PDC.
Magnesium binds to ATP more tightly than ADP.
The amount of free magnesium is reflective of the energy status of the cell.
When ATP is scarce, free magnesium concentrations are higher than when ATP is plentiful.

Arsenic Poisoning

Arsenic inhibits the PDH.
Organic arsenicals were once used to treat syphilis and trypanosomiasis because these organisms’ lipoic acids are more sensitive to arsenic than human homologs.

The Citric Acid Cycle: A Brief History

Krebs proposes the Urea – Ornithine Cycle, introducing the concepts that metabolites can act as catalysis in a cyclic pathway (1932).
Discovery organic carboxylic acids such as citrate, fumarate, oxalic acid, and succinate could, in the presence of oxygen, produce $CO_2$ .
Discovery by Albert Szent-Gyorgyi that the addition of 4 carbon dicarboxylic acids such as succinate, fumarate or oxaloacetate to muscle tissues resulted in the consumption of larger quantities of oxygen required for complete oxidation of the organic acids which indicated that the organic acids were acting as a catalyst for the oxidation of another substrate.
Discovery by Krebs and Johnson that the 6-carbon citrate and 5 carbon α-ketoglutarate also acted catalytically, which led to the suggestion that citrate was formed from a 4-C intermediate and a 2-C derivative of glucose. They also demonstrated that malonate , a succinate dehydrogenase inhibitor, causes accumulation of citric acid cycle intermediates. The citric acid cycle was proposed in 1937.

Citric Acid Cycle Overview

Role: Harvest electrons from acetyl-coA, which can later be used to power metabolic processes.
Two phases: Oxidative (electrons are extracted) and Regenerative (original state is re-established).
Chemical: Oxidation of 2 carbon acetyl-coA to recover energy-rich electrons (8/glucose).
Metabolic: Source of metabolic intermediates for biochemical pathways.

Citric Acid Cycle: A Metabolon

Not the only way to extract electrons from acetyl-coA, but the biologically relevant mechanism.
Lies at the hub of multiple biochemical pathways.
The intermediates of the CAC are also important for other pathways.
Many components of the CAC are present in anaerobic organisms, predating the complete cycle.
Citric Acid Cycle is a metabolon because diffusion of metabolic intermediates through the viscous matrix is inefficient so the enzymes are organized with extensive substrate channeling between active sites.

Citric Acid Cycle: Why?

Very difficult to oxidize a 2 Carbon compound directly and extract the energy and electrons.
Citric acid (6 carbon compound) is easy to oxidize.
Make citric acid by taking 2 Carbon compound (acetyl-coA) and adding it to a 4 carbon compound (oxalic acid).
Oxidize 6 carbon compound easily, 2 times, making 1st a 5 carbon compound, then a 4 carbon compound.
After we oxidize the 6 carbon compound 2 times, we have a 4 carbon compound again.
Turn 4 carbon compound (succinate) back into the 4 carbon compound we want, oxalic acid.

Citric Acid Cycle and Pyruvate

The product of glycolysis is 3C pyruvate, NOT the 2C acetyl-coA.
Pyruvate NOT a substrate of the CAC.
Additional steps are needed to convert 3C into a 2C molecule and recover the energy from converting the pyruvate into acetyl-coA, which is the job for the PYRUVATE DEHYDROGENASE COMPLEX.

Reactions of the citric acid cycle

The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as $CO_2$ in the first turn.
Steps 1, 3, and 4 are essentially irreversible in the cell; all other steps are reversible.
The product of step 5 may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst.

I-Citrate Synthase

The acetyl unit is transferred to a pre-existing molecule of oxaloacetate to form citrate.
The reaction is extremely favorable.
This is an aldol reaction.
Large negative free energy is used to compensate for the relatively low concentration of oxaloacetate that is typically present in the mitochondria.
The favorability ( $\Delta G$ ) of a reaction depends on both $\Delta G^o$ and Q.

Citrate Synthase (Details)

Claisen (aldol like) condensation – a proton is first removed to given the enolate form (equivalent to the carbanion) and this attacks the electrophile on the oxaloacetate.
Hydration of the cit-coA is driven forward by the extremely exergonic cleavage of the thioester.
The proton in acetyl –coA is acidic because it's a “high energy” bond due to no resonance, and large, negative free energy due to the hydrolysis of the coA group pulls forward the prior reaction, making the citric acid spin.
Product is prochiral. Mechanism of the prochirality is by attacking one side of the planar carbonyl group.

Citrate Synthase Mechanism

Illustrates the critical importance of ordered, sequential enzyme kinetics and induced fit.
The oxaloacetate must bind first, inducing a conformational change that allows the acetyl-coA to bind.
After the citroyl-coA is formed additional conformational changes permit the hydrolysis, which prevents hydrolysis of the critical acetyl-CoA.

II. Aconitase

Converts the tert-alcohol (citrate) to the sec-alcohol isocitrate.
Large positive free energy.
Illustrates the property of pro-chirality because it can only remove only the fragment of oxaloacetate is oxidized, not the group delivered by the acetyl-CoA.

Aconitase (Structure)

Iron-sulfur center is in red, the citrate molecule in blue.
Three Cys residues of the enzyme bind three iron atoms; the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate (dashed bond).
A basic residue (:B) in the enzyme helps to position the citrate in the active site and acts in both substrate binding and catalysis.
Moonlighting enzyme that acts as an regulator of iron homeostasis when found in the cytoplasm.

Moonlighting Aconitase

Iron is an important nutrient, forming an essential cofactor in numerous enzymes as well as the oxygen transport/storage proteins, but accumulation of excessive iron is detrimental.
IRP bind to IRE in the mRNA of iron transport/storage proteins.
Aconitase has a dual (moonlighting) function as an IRP and is located in the cytoplasm.

Three Point Attachment Model

Only one of four possible –H atoms can be oxidized by actonitase, even though there are equivalent, so the carboxylic acid and the –OH group must both coordinate the iron.
The –OH group must be positioned on the “left” side of the molecule.
Doing so, while simultaneously contacting an –H with the enzyme, positions the substrate such that only one of the four possible –H can be oxidized.

Get Ready to Oxidize!

All of the subsequent chemistry will be taking place on the portion of isocitrate derived from oxaloacetate.
The atoms derived from acetyl-coA are not modified through a single pass in the citric acid cycle.

Aconitase: The Facts

A moonlighting enzyme that exists in two separate non-exchangeable compartments (cytosol and mitochondria).
Uses an Fe-S cluster and catalyzes a dehydration and a hydration.
The water molecule removed is not the same one that is subsequently added.
Stereospecific coordination of a non-chiral substrate through three points of specific contact.
Converts a prochiral substrate to a chiral product (rehydration is stereospecific).

Fluoroacetate

Can enter into the citric acid cycle as fluoroacetyl-coA and become converted to fluorocitrate.
Fluorocitrate is an inhibitor of aconitase.
Is naturally made by several plant species found in Australia and Africa.
Krebs used Fluoroacetate to study the Citric Acid Cycle because adding it to minced muscle tissue caused an accumulation of CAC intermediates due to termination of the cycle.

III. Isocitrate Dehydrogenase

Substrate, isocitrate, loses one carbon by oxidative decarboxylation.
The electron acceptor can be $NAD^+$ or $NADP^+$ , depending upon the enzyme used.
Is the first energy-recovering step of the citric acid cycle because it is with the NAD+ pathway.
The loss of $CO_2$ driving the synthesis of of an enolate which tautomerizes to the keto.
The $CO_2$ that is lost comes from the pre-existing ‘oxaolacetate’ atoms but no carbons have been lost yet from the original acetyl-coA.
Formation of the carbonyl group enables the oxidative decarboxylation reaction to occur.
CO2 that has been lost was derived from oxaloacetate, not acetyl-coA Note the enol – keto tautomirization, while this reaction is very exergonic and “pulls” the aconitase reaction in the forward direction.

Isocitrate Dehydrogenase (Details)

The isocitrate dehydrogenase reaction can be broken down into two discrete steps.
First, the carbon will be oxidized (by NADP+, as shown here, or NAD+) to give the carbonyl group, forming oxalosuccinate.
Next, the carboxylic acid group on the 3-C is lost (decarboxylation), made possible by the presence of the newly formed carbonyl group.

α-Ketoglutarate Dehydrogenase

The structure and mechanism of this enzyme complex are essentially the same as the Pyruvate Dehydrogenase Complex.
Note the high energy thioester bond.
Using TPP to “sink” the electrons until we can complete the decarboxylation reaction.

α-Ketoglutarate Dehydrogenase Mechanism

Mechanism is entirely analogous to the pyruvate dehydrogenase complex mechanism.

Decarboxylation Comparisons

In the decarboxylation of isocitrate, we can “use” the nearby –OH group to generate a carbonyl group which can subsequently form a resonance stabilized enolate.
No such –OH group exists in alpha keto glutarate, requiring the use of TPP for the decarboxylation.

Succinyl-CoA Synthetase

Molecule has a large amount of chemical potential energy due to the thioester bond so this is the ONLY step that yields ATP (or GTP equivalent).
The net yield of NTP from the citric acid cycle is 1 per turn (2 per glucose).
Energy to drive NTP formation is from the favorable hydrolysis of the thioester this is a substrate level phosphorylation (the only one in the CAC).
Succinyl-CoA Synthetase can be converted to ATP without any energy cost and is also known as succinate thiokinase.

Thioesters and ATP Synthesis

Succinyl-coA we have a thioester, a ”high-energy” compound due to the inability of the carbonyl group to undergo resonance, “trapping” the electrons in the carbonyl group.
A dehydrogenase is oxidizing a substrate and “trapping” the resulting molecule as a thioester.
A phosphate group can attack the thioester to make a phosphoester which can, in turn, transfer the phosphate group down the phosphotransfer potential hill to make an NTP.

Glyceraldehyde-3-Phosphate Dehydrogenase

Examine the second phase of this reaction – where a phosphate group attacks the thioester.
Very similar to what will happen in the citric acid cycle- however, in the CAC, the thioester will be with coA, and not a cysteine in the enzyme.

Phosphoglycerate Kinase

In glycolysis, the phosphoester generated by attack of the phosphate group on the thioester transfers the phosphate down the phosphotransfer potential hill to make ATP - this is substrate level phosphorylation.

α-Ketoglutarate Dehydrogenase (Repeat)

Substrate is becoming oxidized and trapped as a thioester.

Succinyl-CoA Synthetase (Details)

Mechanism: phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. (2) the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. (3) the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP).
$GTP + ADP \rightleftharpoons GDP + ATP, \Delta G^o’ = 0kJ/mol$ ; Nucletotide diphosphate kinase.
Succinyl-CoA synthetase is an αβ heterodimer with the b-subunit determining whether the substrate is ADP or GDP; metabolically active tissues use GDP.

Succinyl-CoA Synthetase (Active Site)

Active site includes part of both the $α$ (blue) and the $β$ (brown) subunits.
The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of P–His246 in the α chain, stabilizing the phosphohistidyl enzyme.
The bacterial and mammalian enzymes have similar amino acid sequences and three-dimensional structures.

Succinate Dehydrogenase

Working towards regenerating the oxaloacetate, through a three step process starting with succinate being oxidized to fumarate.
FADH2 does not leave the enzyme to which it is associated, unlike NADH.
Also known as Complex II, it is the only enzyme of the CAC embedded in the inner mitochondrial membrane.
Inhibition by malonate leads to an accumulation of all CAC intermediates prior to fumarate.
Passing electrons to FAD, not NAD+ which then must be passed to quinone.
Due to mutations in Citric Acid Cycle enzymes, a number of inherited conditions are associated. These include paragangliomas (benign head and neck tumors) as well as Encephalomyopathy (headaches, seizures, strokes, anorexia, and lactic acidosis).
Unusual stereoselectivity.

Fumarase

Hydrates fumarate to give malate.
The reaction is near equilibrium.
Remarkably stereospecific to D-malate.
Fumarase installs an –OH that can be oxidized to recover electrons.

Malate Dehydrogenase

The concentration of oxaloacetate in the cell is extremely low, due to the favorable reaction that occurs in the subsequent step of the cycle and drives rxn forward (Q, again).
Generating another carbonyl group but not using it immediately for a decarboxylation.
Large positive free energy and also functions in gluconeogenesis, as well has having the same role as ornithine in the urea cycle.

The Citric Acid Cycle Summary

After one turn of the cycle, the 2C acetyl group has entered, and two $CO_2$ have left (but note these are NOT the same carbons).
The energy in each step has been conserved by reduction of 3 $NAD^+$ and 1 FAD, as well as the production of 1 ATP.
NO oxygen molecules have yet been consumed, despite this being aerobic metabolism.

$Acetyl-CoA + 3 NAD^+ + FAD+ + GDP + Pi + 2H2O \rightarrow 2CO2 + 3 NADH + FADH2 + GTP + P_i + 2H^+ + CoA$ ; 8 $e^-$

Electron Recovery

NADH is produced by isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and malate dehydrogenase.
FADH2 is produced by succinate dehydrogenase.

Regulation of the Citric Acid Cycle

The exergonic steps are the regulated steps.
Other steps are limited by substrate availability or product excess.

Regulation of Metabolite Flow

The PDH complex is allosterically inhibited when $\frac{[ATP]}{[ADP]}$ , $\frac{[NADH]}{[NAD^+]}$ , and $\frac{[acetyl-CoA]}{[CoA]}$ ratios are high, indicating an energy-sufficient metabolic state. When these ratios decrease, allosteric activation of pyruvate oxidation results.
The rate of flow through the citric acid cycle can be limited by the availability of the citrate synthase substrates, oxaloacetate and acetyl-CoA, or of $NAD^+$ , which is depleted by its conversion to NADH, slowing the three NAD-dependent oxidation steps.
Feedback inhibition by succinyl-CoA, citrate, and ATP also slows the cycle by inhibiting early steps.
Inhibitors of the a-ketogluterate dehydrogenase complex are largely products of the complex.
In muscle tissue, $Ca^{2+}$ signals contraction and, as shown here, stimulates energy-yielding metabolism to replace the ATP consumed by contraction.
Excess citrate (inhibitor) leaves mitochondria for decomposition to oxaloacetate and Acetyl-coA in the cytoplasm , which this is the first level of control to the cytoplasm
In many bacteria, ATP raises the Km of Citrate Synthase.

Citrate as an inhibitor

Appears in the cycle just prior to the two downhill steps.
Can be used to regenerate acetyl-coA and regulate glycolysis therefore, having it act as an inhibitor prior to its entering a thermodynamically downhill reaction is ideal.
In the cytoplasm, citrate is split into acetyl-coA and oxalic acid with an ATP dependent process due to the relatively low concentration of citrate in the cytoplasm.

Citrate Regulates PFK-1

High citrate indicates high downstream metabolic activity (predominantly through fats and protein metabolism) and less requirement for additional F1,6-bisphosphate.

Carbon in the Citric Acid Cycle

The mechanism of the citric acid cycle was established using carbon tracing experiments in which glucose, pyruvate, acetyl co-A or one of the citric acid cycle intermediates was labeled with a carbon isotope and the fate of the isotope after passing through the citric acid cycle was established.
The carbon from the original acetyl-coA have not been lost to one pass through the cycle and the position of the acetyl- coA is “lost” in the conversion of succinyl-coA to succinate. and the position of the acetyl- coA is “lost” in the conversion of succinyl-coA to succinate.

Fate of Carbon Atoms

None of the carbon from the original acetyl-coA have been lost to one pass through the cycle.
Takes two full turns of the cycle for them to be oxidized.

Fate of Carbon Atoms

Takes even longer for the other acetyl-coA carbon to leave the cycle because the first carbons to leave do not do so until the third pass of the cycle, and then a defined fraction leaves thereafter.

Anabolism and Anaplerotic Reactions

The citric acid cycle acts as a central hub for many interconnected metabolic pathways, creating multiple entry points for molecules to be used as fuels and feeds other anabolic pathways, providing intermediates.
Anaplerotic reactions (shown in red) replenish depleted cycle intermediates. Acetyl-coA is directly replaced by breakdown of pyruvate, whereas the reaction we focus on is the carboxylation of pyruvate by pyruvate carboxylase to give oxaloacetate, which Is inactive in the absence of acetyl-CoA.

Pyruvate Carboxylase

Forms the most important anaplerotic reaction, after is has already been seen this reaction in gluconeogenesis. The loss of citric acid cycle intermediates to anabolism results in an accumulation of acetyl-CoA, stimulating to the production of oxaloacetate, regenerating the CAC.
In the cylic acid cycle, the oxaloacetate will not lose the $CO_2$ and form the enolate.

Anaplerotic reactions

Enzymes involved: Malic Enzyme, PEP Carboxylase, and Pyruvate carboxylase.

Amino Acids

Transaminase enzymes generate CAC intermediates from amino acids.
Glutamate dehydrogenase provides a source of a-ketoglutarate from glutamate: $Glutamate + NAD(P)^+ + H2O \rightleftharpoons α-ketoglutarate + NADP(H) + NH4 ^+$

Carbon Skeletons

Amino acids can be converted into one of six different major metabolites: acetyl-coA, pyruvate, oxaloacetate, fumarate, succinyl-coA, and a-ketoglutarate, and each of these molecules can enter into the citric acid cycle and be metabolized to obtain NADH.
Alternatively, the carbon skeletons can enter the CAC and then be diverted to form glucose (glucogeneic amino acids) or ketone bonds (ketogenic amino acids) for fuel.
Some amino acids are both ketogenic and glucogenic due to different parts of their carbon skeletons being metabolized to different intermediates - occurs in the liver involving 7 amino acids in total.

Bookkeeping: Reducing Equivalents

Glycolysis: 2 NADH per glucose (cytosol).
Pyruvate Dehydrogenase Complex: 2 NADH/glucose (mitochondria).
Citric Acid Cycle: 6 NADH/glucose + 2 FADH2/glucose (mitochondria).
Total: 10 NADH (2e-/each) + 2FADH2 (2 e-/each) = 24 electrons.

Bookkeeping: NTPs

Glycolysis: 2 NTP/glucose (cytosol).
Pyruvate Dehydrogenase Complex: 0 NTP/Glucose.
Citric Acid Cycle: 2 NTP/glucose (mitochondria).
Total: 2 NTP (cytosol) + 2 NTP (mitochondria) = 4 NTP.

Glyoxalate Cycle (Krebs III)

Variant of the citric acid cycle that occurs in bacteria, plants, some fungi, algae, and protozoans. In plants cycle happens in a separate organelle called the glyoxome, although in some organisms, steps may also occur in the mitochondria.
It permits organisms to survive on 2 carbon compounds (for example generate glucose from acetyl-coA).
First two reactions are as in the citric acid cycle but the steps leading to the oxidation are skipped so no carbons have been lost as a result of the cycle, since these are the only oxidation steps in the CAC where $CO_2$ is produced.
Isocitrate split into a 4C molecule, and a 2C molecule, glyoxylate:
4 C molecule: lost
2 C molecule: created, along with a
Another Acetyl-coA enters cycle at the box marked in blue. At this moment, we have a 2C molecule and a 4C molecule. it islinked together with the glyoxylate to produce a malate, allowing the cycle to persist
Succinate that was split off from the isocitrate will be continues through the cycle which and it replentishes malate lost to make oxaloacetate and PEP
The is bypass of the oxidation steps is what is key because these enzymes have become co