Table of Contents
After glycolysis has broken down glucose into two molecules of pyruvate, the cell must further process pyruvate before it can enter the citric acid (Krebs) cycle. This “link” between glycolysis and the citric acid cycle consists of two closely connected steps:
- Transport of pyruvate into the mitochondrial matrix (in eukaryotes)
- Conversion of pyruvate into acetyl-CoA by the pyruvate dehydrogenase complex
This stage is often called the “link reaction” or “oxidative decarboxylation of pyruvate”.
Location and Transport of Pyruvate
Different locations in prokaryotes and eukaryotes
- Eukaryotic cells:
- Glycolysis occurs in the cytosol.
- The citric acid cycle and the following electron transport chain occur in the mitochondrion.
- Therefore, pyruvate produced in the cytosol must be transported into the mitochondrial matrix.
- Prokaryotic cells:
- There are no mitochondria; glycolysis and the citric acid cycle occur in the cytosol or associated with the plasma membrane.
- Pyruvate does not need to cross an organelle membrane, but it is still converted into acetyl-CoA by a similar enzyme complex.
The transport process described below refers specifically to eukaryotes.
Crossing the mitochondrial membranes
The mitochondrion has two membranes:
- Outer mitochondrial membrane
- Inner mitochondrial membrane
Pyruvate must cross both to reach the matrix, where the citric acid cycle enzymes are located.
- Outer membrane:
- Contains large, non-selective channels called porins.
- Small molecules like pyruvate diffuse through these channels easily.
- Inner membrane:
- Much more selective and less permeable.
- Pyruvate enters the matrix via a specific transport protein, the mitochondrial pyruvate carrier (MPC).
- Transport is usually coupled to the proton gradient generated by the electron transport chain (an example of secondary active transport), but the exact mechanism can vary between organisms and tissues.
This transport ensures that pyruvate arrives where the next enzyme complex is located: the pyruvate dehydrogenase complex in the matrix.
Overview of Pyruvate Conversion to Acetyl-CoA
Once in the matrix, each pyruvate molecule (3 carbon atoms) is converted to:
- 1 molecule of acetyl-CoA (2 carbon atoms)
- 1 molecule of CO₂
- 1 molecule of NADH + H⁺
This reaction is:
- An oxidative decarboxylation:
- Decarboxylation: removal of a carboxyl group as CO₂
- Oxidation: electrons are removed from pyruvate and transferred to NAD⁺, forming NADH
- Irreversible under cellular conditions
- A key regulatory point between glycolysis and the citric acid cycle
The overall reaction for one pyruvate is:
$$
\text{Pyruvate} + \text{CoA-SH} + \text{NAD}^+
\rightarrow \text{Acetyl-CoA} + \text{CO}_2 + \text{NADH} + \text{H}^+
$$
Because each glucose molecule yields two pyruvate molecules, the conversion per glucose is:
- 2 acetyl-CoA
- 2 CO₂
- 2 NADH + 2 H⁺
The Pyruvate Dehydrogenase Complex (PDC)
A multienzyme complex
The conversion of pyruvate to acetyl-CoA is not carried out by a single enzyme, but by a large multienzyme complex, the pyruvate dehydrogenase complex (PDC).
Key properties:
- Found in the mitochondrial matrix (eukaryotes) or cytosol/plasma membrane region (prokaryotes)
- Consists of three core enzymatic components, often present in multiple copies:
- E1: pyruvate dehydrogenase (decarboxylase)
- E2: dihydrolipoyl transacetylase
- E3: dihydrolipoyl dehydrogenase
- Includes tightly associated coenzymes and prosthetic groups that carry out specific chemical steps
This organization brings all the necessary components together, so intermediates can be passed quickly between active sites (“substrate channeling”).
Required cofactors and coenzymes
The PDC requires several vitamin-derived cofactors:
- Thiamine pyrophosphate (TPP) from vitamin B₁
- Lipoic acid (often covalently attached as a lipoamide arm)
- Coenzyme A (CoA-SH) from vitamin B₅ (pantothenic acid)
- FAD (flavin adenine dinucleotide) from vitamin B₂
- NAD⁺ (nicotinamide adenine dinucleotide) from vitamin B₃ (niacin)
They play different roles in:
- Binding pyruvate
- Transferring acyl groups
- Accepting and donating electrons
A deficiency in the vitamins B₁, B₂, B₃, or B₅ can therefore impair the activity of the pyruvate dehydrogenase complex.
Stepwise Mechanism of Pyruvate to Acetyl-CoA
The overall reaction occurs in a series of tightly coupled steps. Without going into every chemical detail, the key stages are:
Step 1: Decarboxylation of pyruvate (E1, with TPP)
- Pyruvate (3C) is bound by E1.
- A carboxyl group is removed and released as CO₂.
- The remaining 2-carbon fragment (an acetyl group in reduced form) is temporarily attached to TPP on E1.
Result:
- 1 carbon removed as CO₂
- 2-carbon unit bound to TPP (no free acetyl-CoA yet)
- No NADH produced yet
Step 2: Transfer of the acetyl group to lipoamide (E1 → E2)
- The 2-carbon fragment is transferred from TPP (on E1) to the lipoamide arm on E2.
- During this transfer, the fragment is oxidized, and the sulfur atoms of lipoamide become reduced.
Result:
- An acetyl group attached to lipoamide (acetyl-lipoamide)
- TPP is regenerated on E1 for another cycle
Step 3: Formation of acetyl-CoA (E2)
- The acetyl group is transferred from acetyl-lipoamide to CoA-SH (coenzyme A).
- This forms acetyl-CoA, the final product that will enter the citric acid cycle.
Result:
- Free acetyl-CoA
- Reduced lipoamide (dihydrolipoamide), which must be reoxidized to be reused
Step 4: Regeneration of oxidized lipoamide and production of NADH (E3)
- E3 contains FAD as a cofactor.
- Reduced lipoamide is oxidized again by transferring its electrons to FAD, forming FADH₂ and regenerating oxidized lipoamide.
- FADH₂ then transfers the electrons to NAD⁺, forming NADH + H⁺.
Result:
- Oxidized lipoamide restored for another cycle
- Formation of NADH, which carries electrons to the electron transport chain
- Formation of H⁺
Putting all steps together gives the overall equation described above.
Energetic and Metabolic Significance
Why acetyl-CoA is central
Acetyl-CoA is a key metabolic intermediate:
- It enters the citric acid cycle, where its 2-carbon acetyl group is fully oxidized to CO₂.
- It provides a starting unit for fatty acid synthesis, cholesterol synthesis, and other anabolic pathways (in other contexts).
- It acts as a metabolic hub connecting the breakdown of carbohydrates, fatty acids, and some amino acids.
In the context of cellular respiration, its main role is to feed carbon (and high-energy electrons) into the citric acid cycle for further oxidation and ATP production.
Energy output from this step
The conversion of 1 pyruvate produces:
- 1 NADH
Using the approximate ATP yield from oxidative phosphorylation, each NADH can yield roughly 2.5 ATP (depending on the system and exact coupling). Therefore, per glucose:
- 2 pyruvate → 2 NADH
- Approximate contribution: up to 5 ATP equivalents (indirectly, through the electron transport chain)
No ATP is produced directly in this step; the energy is conserved in the form of NADH and the high-energy thioester bond of acetyl-CoA.
Regulation of Pyruvate Dehydrogenase
Because this step determines how much pyruvate enters the citric acid cycle, it is tightly regulated to match the cell’s energy needs.
Allosteric regulation
The pyruvate dehydrogenase complex is inhibited by signals that indicate abundant energy or building blocks:
- Inhibitors:
- High ATP (high energy state)
- High NADH (reduced electron carriers already abundant)
- High acetyl-CoA (citric acid cycle is already well supplied)
- Activators:
- High ADP or AMP (low energy state)
- High pyruvate (substrate availability)
- Low NADH/NAD⁺ ratio
Thus, when energy is plentiful, pyruvate is not funneled into the citric acid cycle as acetyl-CoA; instead, it can be used for other purposes (e.g., fatty acid synthesis, or in other pathways described elsewhere). When energy is needed, more acetyl-CoA is produced.
Covalent modification (phosphorylation/dephosphorylation) in eukaryotes
In many eukaryotic cells, especially in animals, E1 of the complex is additionally controlled by reversible phosphorylation:
- Pyruvate dehydrogenase kinase (PDK):
- Phosphorylates E1 → makes it inactive.
- Activated by high ATP, NADH, acetyl-CoA.
- Pyruvate dehydrogenase phosphatase (PDP):
- Dephosphorylates E1 → makes it active.
- Stimulated by Ca²⁺ and sometimes by insulin in certain tissues.
This covalent regulation allows fine-tuning depending on hormonal signals and tissue type, coordinating carbohydrate utilization with whole-body metabolism.
Alternatives to Acetyl-CoA Formation
When oxygen is limited or when mitochondrial function is impaired, pyruvate may:
- Be reduced to lactate (e.g., in muscle) or converted via other fermentation pathways (covered under fermentation).
- Be used in other metabolic routes (e.g., gluconeogenesis in some tissues).
In these situations, the pyruvate dehydrogenase reaction is reduced or bypassed, and less acetyl-CoA is formed for the citric acid cycle. This illustrates that conversion of pyruvate to acetyl-CoA is not only a chemical transformation, but also a regulated branching point in metabolism.
Summary
- Pyruvate produced by glycolysis must reach the mitochondrial matrix (in eukaryotes) via the mitochondrial pyruvate carrier.
- In the matrix, the pyruvate dehydrogenase complex (PDC) converts pyruvate into acetyl-CoA, CO₂, and NADH.
- This reaction is an oxidative decarboxylation, is effectively irreversible, and serves as a key link between glycolysis and the citric acid cycle.
- The PDC is a large multienzyme complex requiring several vitamin-derived cofactors and is tightly controlled by both allosteric effectors and (in many eukaryotes) phosphorylation–dephosphorylation.
- The acetyl-CoA formed is a central metabolite feeding the citric acid cycle and many other pathways, making this step crucial for the control and integration of cellular energy metabolism.