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16.1: Production of Acetyl-CoA (Activated Acetate)

  • Page ID
    15018
  • Introduction

    Let's do a short review of the metabolic processes to the extraction of energy from the oxidation of glucose (i.e. glycolysis). Glycolyis, a universal pathway used to anaerobically extract energy from glucose, a six carbon sugar. 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 endproduct of glycolysis, can be converted to lactate, regenerating NAD+ so the pathway can continue.

     

    PreTCA_GlycolysisOverview.svg

     

    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 the figure below.

    alphaketovsbetaketoaciddecarb.svg

    To begin the process of complete oxidation of the remnants of glucose, pyruvate enters the mitochondria and starts the process of oxidative decarboxlations by interacting with the pyruvate dehydrogenase complex (PHC). This catalyzes an extremely complicated set of reaction to attach an electron "sink" beta to the carboxlate, which is subsequently released as CO2. The end products of the PHC oxidative decarboxylation reaction are the two carbon acetyl-CoA molecule, NADH and CO2. The acetyl-CoA then enters a cyclic, non-linear pathways called the citric acid cycle, tricarboxylic acid (TCA) cycle or the Kreb's cycle after Hans Krebs who discovered it.  We'll talk about that in sections 16:2.  

    A quick glance reveals that we have taken glucose a small fraction along the way of oxidizing every carbon in it to CO2 and H2O. The complete oxidation happens under aerobic condition when the glycolytic pathway is followed by the Kreb's cycle. Pyruvate formed in glycolysis enters the mitochondrial matrix, and get oxidatively decarboxylated while reacting with a small thiol, Coenzyme A (CoASH) to form a 2C "activated acetate" acetyl group connected through a thiolester link to CoASH, forming acetylCoA.    

    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 enyzmes 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 a histidine and aspartate to cleave peptide bonds.  There are three α-ketoacid dehydrogenases complexes in many organisms.  Each have a common E3 but specific E1 and E2 enzymes.  The image below shows an image of the structure so you can get an overview before we dive into the activity of each of the substrates. The E3 subunit are not readily seen on the image below.  Why such as 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.

    https://electron.med.ubc.ca/2018/07/...dehydrogenase/

    https://upload.wikimedia.org/wikiped...se_complex.jpg 

     

    PyruvateDehydrogenaseComplex_CryoPS1.svg

     

     

    The complex also employees 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 lysine side chain in E2
    • riboflavin the 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 the figure below, along with some additional descriptions that summarize some of the chemistry of these molecules.  

     

    VitaminsPyruvateDehydrogenaseComplex.svg

    Here is a schematic of the overall reaction.

     

    metabolismWP_PyruvateDehydroABC_Summary.svg

    The net reaction is

    pyruvate + CoASH + NAD+   →    Acetyl-CoA  + CO2 + NADH + H+

    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 carboxlic acid ester.  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 (see Table X below).  This minimizes resonance stabilization of compared to the carboxylic acid ester,as shown in the figure below.  The products of hydrolysis of both types of esters, a carboxylic acid and either an alcohol or thiol, are of comparable energy.  Hence only the thioester is relatively destabilized compared to its hydrolysis product, with the ΔG0 hydrolysis = -7.5 kcal/mol, the same as for the hydrolysis of a phosphoanhydride bond of ATP.  Additionally, a resonance structure show a + charge on the carbonyl C and a - on the oxygen allows 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 need to stabilized the thiol ester.

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

    The figure below illustrates these points.

    EsterThioester.svg

     

    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 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 an ylide, which is a net neutral species with a + charge (usually on a N, P or S) and - charge (usually on a C) on adjacent atoms. 

    The carbanion on the ylid 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 enyzme to its original state (E2 and E3).

    The figure below shows the reaction mechanism of E1.

    metabolismWP_PyruvateDehydroA_100520.svg

    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 a 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 attacedh 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 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 acteyl phosphinate, a TPP analog, covalently attached (PDB ID: 6CFO).  One E1 subunit is actually 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.

    https://structure.ncbi.nlm.nih.gov/i...QHhpmTogPMvGF7 

     

    Here is the covalently bond phosphonate TPP analog

     

    https://structure.ncbi.nlm.nih.gov/i...xgmV7rnPirqf4A

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

    keyIMFlines-01.svg

    Exercise \(\PageIndex{1}\)

    Add exercises text here.

    Answer

    Add texts here. Do not delete this text first.

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

    The next part of the reaction produce 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 reduce) in another round of catalysis.  To regenerate enyzme activity, the reduced lipoamide, after release of the attached acetyl group, must be reoxidized by another oxidizing agent.  That oxidizing agent is FAD, which is covalently attached to E3, 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). 

    metabolismWP_PyruvateDehydroB_AloneE2.svg

     

     

    The reaction of E3 follows to restore the fully catalytic enzyme.

     

    metabolismWP_PyruvateDehydroCAlone_E3.svg

     

     

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

    E2: dihydrolipoyl acetyltransferase  -

    In the mammalian complex, 60 E2 subunit arranges into a pentagonal dodecahedron.  Most gram-negative bacteria E2 subunits arranges into a cubic of 24 monomers. Here is simple view of a pentagonal dodecadron, which has 12 equivalent faces..

    120px-Dodecahedron.gif

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

    The E2 inner core 60 mer is shown in the iCn3D model below (pdb 6CT0) .  Symmetry axes are also shown.

    https://structure.ncbi.nlm.nih.gov/i...FED6Q3wZxMynB7

    https://www.ncbi.nlm.nih.gov/Structu...11&command=set background white; symmetry I (global)|||{"factor":"1.000","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"-0.009396","_y":"-0.02119","_z":"-0.0004849","_w":"0.9997"}}

     

    To see the individual monomer, go tot the = menu command (top left of the modeling window) and select Analysis, Assembly, Asymmetric Unit

    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 derives 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 in 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 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 enyzme total competent for another round of catalysis.  DOI : 10.1002/chem.201704622

    The iCn3D model below shows the structure of E3 bound to both FAD (noncovalently) and NADH (NAI in the model)

     

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?CwAAq52YmBEMcZkHA

    The model below shows the interactions that stabilizes the bound forms of both NADH and FAD.

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?jL7Jh5afzTsi4T1L9

    Let's put it all together

     

     Here is a video of the pyruvate dehydrogenase complex - permission uncldar

    Summary

     

    End of Chapter Problems

    Zach

    Exercise \(\PageIndex{1}\)

    What amino acids is named after Trump.

    Answer

    None

    References

    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

    2. 

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