16.1: Production of Acetyl-CoA (Activated Acetate)

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Learning Goals (ChatGPT o3-mini)

1. Understand the Role of PDC in Metabolism:

   • Explain how the pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA, linking glycolysis to the Krebs cycle and overall aerobic respiration.

2. Describe the Structural Organization of PDC:

   • Identify the three major enzymatic components of the PDC (E1, E2, and E3) and describe their subunit composition and overall architecture (e.g., E1 as an α₂β₂ heterodimer, E2 forming a large oligomeric core, and E3 as a smaller, tightly-bound enzyme).

3. Detail the Reaction Mechanism of the E1 Component:

   • Explain the mechanism of oxidative decarboxylation catalyzed by pyruvate dehydrogenase (E1), including the formation of the TPP-ylide intermediate, nucleophilic attack on pyruvate’s carbonyl carbon, and the role of allosteric stabilization in enabling decarboxylation.

4. Examine the Role of Cofactors in PDC Catalysis:

   • List and describe the five essential cofactors involved in PDC activity (TPP, lipoic acid/lipoamide, Coenzyme A, FAD, and NAD⁺), including their chemical structures, sources (e.g., thiamine, riboflavin, niacin, and pantothenic acid), and how they contribute to the reaction mechanism.

5. Compare Thioester and Ester Chemistry:

   • Analyze the differences in resonance stabilization and bond lengths between carbon-oxygen and carbon-sulfur bonds, and explain why thioesters (like acetyl-CoA) are considered “high-energy” intermediates in metabolism.

6. Detail the E2 Catalyzed Transacetylation Reaction:

   • Describe how the dihydrolipoyl acetyltransferase (E2) transfers the acetyl group from the TPP intermediate to Coenzyme A, forming acetyl-CoA, and discuss the role of the lipoamide “swinging arm” in facilitating substrate channeling within the complex.

7. Understand the E3 Reaction and Enzyme Regeneration:

   • Explain the function of dihydrolipoyl dehydrogenase (E3) in reoxidizing the reduced lipoamide via FAD and NAD⁺, thereby regenerating the oxidized cofactor necessary for continuous PDC activity.

8. Utilize Structural Models to Visualize PDC Components:

   • Engage with interactive iCn3D models (e.g., PDB IDs 6CFO, 1ZMD, 6CT0) to identify key active site residues, cofactor binding sites, and structural domains in E1, E2, and E3, enhancing understanding of how structure relates to function.

9. Integrate Multistep Reaction Mechanisms:

   • Synthesize the sequential reactions from pyruvate decarboxylation, acetyl group transfer, and cofactor regeneration to explain the overall net reaction catalyzed by the pyruvate dehydrogenase complex.

10. Discuss the Biological Significance of PDC Regulation:

    • Evaluate why nature has evolved such a large and complex enzyme system for the conversion of pyruvate to acetyl-CoA, considering factors such as substrate channeling, regulation by cofactors, and the integration of multiple catalytic activities to ensure efficient energy production.

These learning goals aim to guide students through both the mechanistic intricacies and the structural organization of the pyruvate dehydrogenase complex, linking detailed biochemical principles with broader metabolic contexts and energy homeostasis.

Introduction

Let's briefly review the metabolic processes for energy extraction from glucose oxidation (i.e., glycolysis). Glycolysis is a universal pathway to extract energy from glucose anaerobically. In this linear pathway, 2-3 carbon molecules of pyruvate are formed as glucose is cleaved and converted to two molecules of glyceraldehyde-3-phosphate, formed through an oxidation reaction using the oxidizing agent NAD⁺. As glycolysis continues, NADH builds up. Using lactate dehydrogenase, pyruvate, the end product of glycolysis, can be converted to lactate, regenerating NAD⁺ so the pathway can continue. The reactions are illustrated in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Anaerobic production of pyruvate and lactate

A careful glance at the structure of the 3-carbon pyruvate molecule shows that much more energy could be extracted from it, presumably through oxidative decarboxylation reactions, converting the carbons to 3 CO₂ molecules. A problem arises immediately when examining pyruvate. It is an α-ketoacid, and there is no easy route to decarboxylate it as an electron "sink" is not available to receive the electrons and stabilize the transition state and intermediate in the reaction. In contrast, the decarboxylation of β-keto acids has a built-in electron "sink", an electronegative carbonyl carbon, to receive the electrons. This is illustrated in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Comparison of the decarboxylation of α and β keto acids

To begin the complete oxidation of the remnants of glucose, pyruvate enters the mitochondria and starts the process of oxidative decarboxylations by interacting with the pyruvate dehydrogenase complex (PHC). This catalyzes a complicated reaction to attach an electron "sink" beta to the carboxylate, which is subsequently released as CO₂. The end products of the PHC oxidative decarboxylation reaction are the two-carbon acetyl-CoAs, NADH, and CO₂. The acetyl-CoA then enters a cyclic, non-linear pathway called the citric acid cycle, tricarboxylic acid (TCA) cycle, or Krebs cycle, named after Hans Krebs, who discovered it. We'll talk about that in section 16.2.

A glance reveals that we have taken glucose a small fraction towards completely oxidizing every carbon into CO₂ and H₂O. Complete oxidation happens under aerobic conditions in the Krebs cycle. Pyruvate formed in glycolysis enters the mitochondrial matrix where it gets oxidatively decarboxylated while reacting with a small thiol, Coenzyme A (abbreviated either as CoA or CoASH ) to form a 2C "activated acetate" acetyl group connected through a thioester link to CoASH, forming acetyl-CoA.  Coenzyme A is often abbreviated as CoASH to emphasize that it has a free nucleophile thiol (-SH) group.

The third carbon from pyruvate is released as CO₂. The reaction is catalyzed by the enzyme pyruvate dehydrogenase complex (PDC).

Pyruvate Dehydrogenase Mechanism

This enzyme complex is enormous. The E. Coli complex has a molecular weight of almost 4 million with at least 16 chains of three different enzymes catalyzing part of the reaction. The components are pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E2), and dihydrolipoamide dehydrogenase (E3). The molecular weight of the bovine complex is almost 8 million, and it has 22 E₁, 60 E₂, and 6 E₃ subunits. Nature often uses the same solution for identical problems. For example, many proteases have an active site nucleophilic serine, which works with the assistance of histidine and aspartate to cleave peptide bonds. There are three α-ketoacid dehydrogenase complexes in many organisms. Each has a common E3 but specific E1 and E2 enzymes. Figure \(\PageIndex{3}\) shows an image of the structure so you can get an overview before we dive into the activity of each of the substrates.

Figure \(\PageIndex{3}\): View of pyruvate dehydrogenase. https://electron.med.ubc.ca/2018/07/...dehydrogenase/

The E3 subunit is not readily seen in the image above. Why has nature produced such a monstrous enzyme complex simply to catalyze the oxidative decarboxylation of a small three-carbon molecule? We will explore that at the end of this section.

The complex also employs five substrates/cofactors derived from vitamins collectively.

• Thiamine in the form of thiamine pyrophosphate (TPP), which is covalently attached to E1
• lipoic acid, in the form of lipoamide, which is covalently attached to a lysine side chain in E2
• riboflavin in the form of flavin adenine dinucleotide (FAD/FADH₂), which is bound very tightly (and not released) to E3
• pantothenic acid, incorporated into the structure of CoASH/Acetyl-CoA, a substrate/product pair for the reaction
• niacin, nicotinic acid, in the form of NAD⁺/NADH, a substrate/product pair for the reaction

The structures for the five are shown in Figure \(\PageIndex{4}\), along with some additional descriptions that summarize some of the chemistry of these molecules.

Figure \(\PageIndex{4}\): Structure of the cofactors in pyruvate dehydrogenase

Figure \(\PageIndex{5}\) shows a schematic of the overall reaction.

Figure \(\PageIndex{5}\): Overall reactions catalyzed by pyruvate dehydrogenase

The net reaction is

\[\ce{pyruvate + CoASH + NAD^{+} -> Acetyl-CoA + CO2 + NADH + H^{+}} \nonumber \]

Part 1: Oxidative Decarboxylation - pyruvate dehydrogenase (E1p)

So let it begin. We need to eliminate one carbon as CO₂ and transfer the other two carbons of pyruvate to CoASH to form acetyl-CoA, the thioester of CoASH. Thioesters are "high energy" with respect to their hydrolysis products as the thioester is destabilized compared to a normal carboxylic acid ester.  (Remember, there is no such thing as a "high energy" bond.) Since the sulfur atom is larger than the O in the C-S and C-O bond in their respective esters, the thioester as a reactant can not be stabilized well as the C-S single bond length is longer, as shown in Table \(\PageIndex{1}\) below.

bond	length (Angstroms)
C-O	1.43
C=O	1.21
C-S	1.82
C=S	1.56

Table \(\PageIndex{1}\): Bond lengths of carbon-oxygen and carbon-sulfur single and double bonds

This minimizes resonance stabilization compared to the carboxylic acid ester, as shown in the figure below. The hydrolysis products of both a carboxylic and thiol ester are of comparable energy. Hence, only the thioester is relatively destabilized compared to its hydrolysis product, with the ΔG⁰ _hydrolysis = -7.5 kcal/mol (-31 kJ/mol), the same as for the hydrolysis of a phosphoanhydride bond of ATP. Additionally, a resonance structure shows a positive charge on the carbonyl C and a negative charge on the oxygen, allowing the carbonyl carbon to be more electrophilic. Another more sophisticated reason for the relative destabilization of the thiol ester is that the overlap between the carbonyl C p orbital is the larger S p orbital is less, hindering the delocalization of electrons needed to stabilize the thiol ester.

Figure \(\PageIndex{6}\) illustrates these points.

Figure \(\PageIndex{6}\): Comparison of resonance stabilization of carboxylic esters and thioesters

Now, we can explore the mechanism of CO₂ release and acetyl-CoA production by E₁. The carbon atom directly between the N and S in the thiamine ring has a reduced pKa, so it can be deprotonated to form a carbanion. The negative charge can't be stabilized by resonance but is adjacent to the positively charged N, which does. This zwitterion is called a ylide, a net neutral species with a positive charge (usually on a N, P, or S) and a negative charge (usually on a C) on adjacent atoms.

The carbanion on the ylide attacks the electrophilic C=O of pyruvate, forming a TPP intermediate with an excellent electron sink (N⁺) beta to the carboxyl carbonyl C. This is the essence of the entire reaction as this enables the decarboxylation event. The rest of the reactions catalyzed by E2 and E3 allow the release of the other 2 Cs of pyruvate as acetyl-CoA (E2) and the return of the enzyme to its original state (E2 and E3).

Figure \(\PageIndex{7}\) shows the reaction mechanism of E1.

Figure \(\PageIndex{7}\): Reaction mechanism of E1 of pyruvate dehydrogenase

Note that the carbonyl C in pyruvate has two single bonds to two other carbon atoms, while in the final covalently attached form, it has one bond to carbon and one to sulfur. Sulfur is under oxygen in the periodic table. By analogy, replacing one C-C bond with a C-S bond is an oxidation reaction, which requires an oxidizing agent. The covalently attached ring of the lipoamide with an S-S bond similar to that of a disulfide bond, an oxidized form of sulfur, is the oxidizing agent. On the formation of acetyl-lipoamide, the S-S bond is cleaved, and a thioester is formed. Other sulfur is a free reduced thiol.

There are 22 E1 subunits in the bovine PDC. Here is an iCn3D model of one human pyruvate dehydrogenase E1 component complex with TPnP (TDP acetyl phosphonate, a TPP analog, covalently attached (PDB ID: 6CFO). One E1 subunit is an α₂β₂ heterodimer. The two alpha chains are cyan, while the beta chains are dark blue. Orient the model to view along the C2 rotational symmetry axes shown.

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the human pyruvate dehydrogenase E1 covalently bound to TDP acetyl phosphonate (TpnP), a TPP analog (6CFO)

Figure \(\PageIndex{8}\): Human pyruvate dehydrogenase E1 covalently bound to TDP acetyl phosphonate (TpnP), a TPP analog (6CFO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...7FJ2GE47tvS6g7

A heterotetramer containing two α (cyan) and two β chains (dark blue) is shown.

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of the TPP analog covalently bound to E1 of pyruvate dehydrogenase (6CFO)

Figure \(\PageIndex{9}\): TPP analog covalently bound to E1 of pyruvate dehydrogenase (6CFO) . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SNf5sC7SqmQkc8

The dotted lines show the interactions between the TPP analog, color-coded as shown in the legend below.

 

Parts 2 and 3: Acetyl-CoA (E2) Formation and Regeneration of the Active Complex.

The next part of the reaction produces acetyl-CoA (E2).  After that, the enzyme is "dead" as it no longer has an oxidized form of lipoamide to serve as an oxidizing agent (which gets reduced) in another round of catalysis. To regenerate enzyme activity, the reduced lipoamide, after the release of the attached acetyl group, must be reoxidized by another oxidizing agent. That oxidizing agent is FAD, which is covalently attached to E3 and is converted to FADH₂. It must be reoxidized back to FAD to restore activity to the enzyme complex. The final oxidizing agent used for that is solution-phase NAD⁺, which the enzyme releases as a product. So it's a bit complicated. Three oxidizing agents are used in the PDH.  Two are covalently attached to the enzyme (oxidized lipoamide on E2 and oxidized FAD on E3).

Figure \(\PageIndex{10}\) shows the transacetylation reaction and formation of reduced lipoamide

Figure \(\PageIndex{10}\): Transacetylation reaction and formation of reduced lipoamide by pyruvate dehydrogenase E2

The reaction of E3 follows to restore the fully catalytic enzyme, as shown in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Regeneration of oxidized lipoamide by pyruvate dehydrogenase E3.

Let's look in greater detail at the structures of both E2 and E3.

E2: dihydrolipoyl acetyltransferase -

In the mammalian complex, 60 E2 subunits arrange into a pentagonal dodecahedron. Most gram-negative bacteria E2 subunits arrange into a cube of 24 monomers. Figure \(\PageIndex{12}\) shows a simple view of a pentagonal dodecahedron with 12 equivalent faces.

Figure \(\PageIndex{12}\): Pentagonal dodecahedron. 1. Rotating dodecahedron: https://commons.wikimedia.org/wiki/F...decahedron.gif. User Cyp on en.Wikipedia, CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons

First, let's consider one single E2 monomer. It has a longer disulfide redox domain followed by a smaller dimerization domain, which allows the assembly of multiple subunits into the dodecahedron. In greater detail, the monomer has two lipoyl domains, a small domain that allows binding to E1 and a C-terminal catalytic domain.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of E2 inner core 60-mer of human pyruvate dehydrogenase (pdb 6CT0). Symmetry axes are not shown

Figure \(\PageIndex{12}\): E2 inner core 60-mer of human pyruvate dehydrogenase (pdb 6CT0). (Copyright; author via source). Click the image for a popup or use this external: https://structure.ncbi.nlm.nih.gov/i...zs6pwfKNuJ2VS6

Each of the 60 subunits is shown in light cyan. To see the C symmetry axes:

• select the menu =
• Choose/Check in order: Analysis, Symmetry, From PDB, 1(global), apply
• When you see just a single monomeric chain choose Clear

The symmetry axes will then appear.

E3: dihydrolipoyl dehydrogenase

The sole function of this subunit is the reoxidation of the now-reduced lipoamide with the free sulfhydryl to the cyclic disulfide form so the enzyme can engage in further catalysis. FAD covalently bound to the E3 subunit is the oxidizing agent. This is our first encounter with FAD. Similarly to NAD+, this dinucleotide gains a hydride (:H-) but also, in contrast to NAD⁺, also a proton to form FADH₂.

Another way that the FAD/FADH₂ differs from NAD⁺/NADH is that the FAD/FADH₂ or their mononucleotide analog (FMN/FMNH₂) pairs are either covalently attached (in about 10% of flavoproteins) or bound with such a low K_D (often in the nanomolar range) that they don't dissociate from the enzyme during catalysis. Hence, after oxidizing a bound substrate, the reduced FADH₂ must be reoxidized by another oxidizing agent, often NAD⁺, which can diffuse into the active site to do its job and then dissociate from the complex in the form of NADH, leaving the enzyme competent for another round of catalysis. (DOI: 10.1002/chem.201704622)

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of E3 bound to both FAD (noncovalently) and NADH (NAI) (1ZMD) in the B chain of E3. Symmetry axes are not shown.

Figure \(\PageIndex{13}\): E3 bound to both FAD (noncovalently) and NADH (NAI) (1ZMD). (Copyright; author via source). Click the image for a popup or use this external: https://structure.ncbi.nlm.nih.gov/i...LAMqAS3Tx9SPE8.

Figure \(\PageIndex{14}\) shows an interactive iCn3D model highlighting the noncovalent interactions stabilizing bound FAD and NADH (NAI) in the E3 subunit of pyruvate dehydrogenase (1ZMD).

Figure \(\PageIndex{14}\): Noncovalent interactions stabilizing bound FAD and NADH (NAI) in the E3 subunit of pyruvate dehydrogenase (1ZMD). (Copyright; author via source). Click the image for a popup or use this external:https://structure.ncbi.nlm.nih.gov/i...gUED1ZrJVjKcD8

Let's put it all together! Figure \(\PageIndex{15}\) shows a video of the pyruvate dehydrogenase complex from the HHMI.

Figure \(\PageIndex{15}\): Video of the pyruvate dehydrogenase complex 

Summary

This chapter provides an in-depth look at how cells extract energy from glucose beyond glycolysis by focusing on the pyruvate dehydrogenase complex (PDC), a critical enzyme system that links glycolysis to the citric acid cycle. Key topics include:

• From Glycolysis to Acetyl-CoA:
  Glycolysis converts glucose into pyruvate, a three-carbon molecule. Although glycolysis is efficient for anaerobic energy extraction, pyruvate can be further oxidized under aerobic conditions. The chapter explains that the direct oxidative decarboxylation of pyruvate is challenging due to its α-ketoacid structure lacking a built-in electron sink. PDC overcomes this limitation by using a series of cofactors to facilitate decarboxylation and the formation of acetyl-CoA.

• Structural Organization of the Pyruvate Dehydrogenase Complex:
  PDC is a massive multienzyme complex composed of three key components:

  • E1 (Pyruvate Dehydrogenase): Functions as an α₂β₂ heterodimer that uses thiamine pyrophosphate (TPP) to form a reactive ylide intermediate, enabling the decarboxylation of pyruvate.
  • E2 (Dihydrolipoyl Acetyltransferase): Transfers the acetyl group from the TPP intermediate to Coenzyme A (CoA) via a “swinging arm” mechanism using lipoamide as a carrier.
  • E3 (Dihydrolipoyl Dehydrogenase): Reoxidizes the reduced lipoamide using FAD and NAD⁺, regenerating the oxidized form necessary for continued catalysis.

• Cofactor Utilization and Chemical Basis:
  The complex requires five key cofactors, each derived from essential vitamins:

  • Thiamine pyrophosphate (TPP) – from vitamin B₁
  • Lipoic acid (as lipoamide) – critical for substrate channeling
  • Coenzyme A (CoA) – derived from pantothenic acid
  • Flavin adenine dinucleotide (FAD) – from riboflavin
  • Nicotinamide adenine dinucleotide (NAD⁺) – from niacin
    These cofactors orchestrate a sequence of redox reactions that not only decarboxylate pyruvate but also generate high-energy thioester bonds in acetyl-CoA, an intermediate vital for entry into the citric acid cycle.

• Mechanistic Insights:
  Detailed discussion is provided on the reaction mechanism of E1, emphasizing the formation of a TPP-bound ylide that initiates nucleophilic attack on pyruvate. The subsequent steps involve transacetylation (mediated by E2) and the reoxidation of lipoamide (mediated by E3), underscoring how electron transfer and decarboxylation are tightly coupled within the complex. The chapter also highlights the chemical rationale behind the “high-energy” nature of thioester bonds in acetyl-CoA, drawing comparisons with oxygen-based esters.

• Structural Visualization:
  The chapter incorporates interactive structural models (iCn3D) of PDC components, allowing students to explore the spatial arrangement of active sites, cofactor binding domains, and subunit organization. These models help illustrate how the large, multi-subunit architecture of PDC facilitates efficient substrate channeling and coordinated catalysis.

• Biological Significance:
  Finally, the chapter discusses why such a complex enzyme system is necessary for cellular energy metabolism. The PDC not only ensures the efficient conversion of pyruvate to acetyl-CoA, but its regulation through multiple cofactors and subunit interactions also allows fine control over metabolic flux into the citric acid cycle, ultimately influencing ATP production and overall energy homeostasis.

In summary, this chapter bridges detailed enzymatic mechanisms with broader metabolic integration, offering students a comprehensive understanding of how the pyruvate dehydrogenase complex serves as a critical nexus between anaerobic glycolysis and aerobic respiration.