16.1: Production of Acetyl-CoA (Activated Acetate)
- Page ID
- 15018
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Let's do a short review of the metabolic processes for the extraction of energy from the oxidation of glucose (i.e. glycolysis). Glycolysis is a universal pathway used to anaerobically extract energy from glucose, a six-carbon. 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, form 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 in the process stabilize the transition state and intermediate in the reaction. This stands in contrast to the decarboxylation of β-keto acids, which have 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 process of 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 along the way of oxidizing every carbon in it to CO2 and H2O. Complete oxidation happens under aerobic conditions when the glycolytic pathway is followed by 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 each 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 to simply catalyze the oxidative decarboxylation of a small three-carbon molecule? We will explore that at the end of this section.
The complex also employs collectively 5 substrates/cofactors derived from vitamins.
- 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 get rid of 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 products of hydrolysis 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 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 it is adjacent to the positively-charge N, which stabilizes it. This zwitterion is called a ylide, which is 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 a wonderful 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 so by analogy, the replacement of 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 that has TPnP (TDP acetyl phosphonate, a TPP analog, covalently attached (PDB ID: 6CFO). One E1 subunit is an α2β2 heterodimer. The two alpha chains are shown in cyan while the beta chains are in 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 2 α (cyan) and 2 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: Formation of Acetyl-CoA (E2) and Regeneration of the Active Complex.
The next part of the reaction produces acetyl-CoA (E2), but 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 is released by the enzyme as a product. So it's a bit complicated. Three oxidizing agents are used in the PDH, two of which 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 arranges into a pentagonal dodecahedron. Most gram-negative bacteria E2 subunits arrange into a cubic of 24 monomers. Figure \(\PageIndex{12}\) shows a simple view of a pentagonal dodecahedron, which has 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 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 KD (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