Skip to main content
Biology LibreTexts

16.2: Reactions of the Citric Acid Cycle

  • Page ID
    41618
  •  

    Citric Acid Cycle - aka tricarboxylic acid cycle, TCA cycle or Kreb's cycle)

    Introduction

    The acetyl-CoA formed by the pyruvate dehydrogenase complex (PDC) now 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. The cycle below is shown in wedge/dash form with stereochemistry included to give a more exact representation. 

    Note:  new way to make clear svg images of ChemDraw (or probably Marvin as well)

    1. paste cdx (usally constructed in letter size size) into Word whose default is letter size as well.
    2. save docx as PDF
    3. load into Adobe Illustrator (not Photoshop which can't save as svg)
    4. Select top left Pointer icon and click near image until a box appears around the whole object (not individual components in diagram), then click box to set it 
    5. select artboard icon (looks like crop icon, 2/3 way down on left hand side on top of magnifying glass icon).  Click then resize artboard to same size as total object box from (4)
    6. Select Save, svg and these choices:  SVG Profiles 1.1,  Font - convert to online, Image location - imbed.  

     

     

    TCA_WedgeDashView.cdx_PPTX_LtSize-03.svg

     

    Why is this pathway a cycle and not a linear pathway as we have seen for glycolysis? A simple answer is that it evolved that way, but why would that be advantageous? It turns out the some of the key "intermediates" in the pathway are pulled away for biosynthesis of other biomolecules. If the citric acid cycle was linear, and intermediates pulled off for other reactions, the linear pathway would taper off, which would not be optimal for a key energy production pathway. Of course, removal of intermediates from a cyclic pathway would also slow it down but when this happens, enzymes outside of the cycle are used to produce key reaction intermediates of the cycle to it going. The replenishing reactions are called anapleurotic reactions.

    Krebs, in his detailed analysis of the enzymes involved in "intermediary" metabolism, used radioistope-labeled reactants to trace carbon atoms in respiring tissue. He found that when radiolabeled pyruvate and oxaloacetate were added to muscle tissue in vitro, radiolabeled citrate was formed.

    Pyruvate + Oxaloacetate → citrate + CO2

    This is correct but omits the initial conversion of pyruvate to acetyl-CoA. Hence the "end product" of the pathway (oxalacetate) reforms the beginning reactant (citrate) so he surmised that the pathway was circular.

    The next obvious question is why are eight reactions are required for the oxidation of just 2 Cs in pyuvate. Partly this is a matter of evolution again, as the early evolutionary pathway probably combined an oxidative (clockwise) set of reaction with a reductive (counterclockwise) set. Part of the chemistry in the cycle is devoted to producing either β-keto acids, which are easy to oxidatively decarboxlate, or converting α-ketoacid to molecules with better electron sinks β to the departing CO2. After the net 2 carbon atoms added to the cycle are released as 2 CO2s, the rest of the reactions are used to regenearate oxaloacetate, allowing the cycle to continue.

    Of course, the ultimate goal of an energy-extractive oxidative pathway is not just to form CO2 but to form ATP or its equivalent (i.e. GTP). Notice that 3 NAD+s are used and converted to 3 NADH. In addition, a new, more potent oxidizing agent, FAD, is used and it is converted to FADH2. NAD+ and FAD are replenished by reoxidation of NADH and FADH2 (reduced forms) back to NAD+ and FAD, through mitochondrial electron transport (oxidation) reactions, in which electrons are passed to stronger and stronger oxidizing agents, the last being O2.  In this thermodynamically favored process, lots of ATPs are made. We will explore those reactions in the next secction.

    We will go through each of the steps in the citric acid cycle separately and show how the pathway is regulated (sections 16.3). Why such detail? There are only 8 steps. It seems that we should be able to carefully examine each carefully given that the citric acid cycle is a hub that along with glycolysis controls metabolic flow through many interconnected metabolic pathways.

    At the same time, we can't explore each reaction in every pathway described in this text in great detail, otherwise this book would become more of an encyclopedia. In this chapter and beyond, we will focus on mechanistic details only of enzymes that catalyze different types of reactions than those in glycolysis or the citric acid cycle, and those with interesting cofactors and mechanisms.  

    There are other issues that add complexity for learners. The PDC and citric acid cycle reaction occur in the mitochondrial matrix. Cytoplasmic pyruvate and NAD+ must be transported into the matrix from the cytoplasm. In addition, some of the enzymes in the citric acid cycle have both cytoplasmic and mitochondrial variants. Some of these homologous pairs are differentiated by their use of NAD+ or NADP+ as an oxidizing agent. The ones in the cytoplasm are not part of the cycle. You would expect these enzyme pairs to have similar tertiary structures and active site chemistry. Prokaryotic forms of these enyzmes are similar structurally to their eukaryotic forms so the interactive molecular models shown below will show enzymes from a variety of organisms. Finally, there are many variants, shunts and bypasses of the citric acid pathway in different organisms. We will explore this topic in section 16.4

    We will try to pair reaction mechanism diagrams that show the the flow of electrons in bond making and breaking with interactive models of the active you. You should rotate the models to align and identify key amino acid and ligands (substrate, substrate analogs, inhibitors, activators) shown in the static 2D mechanism diagrams. Since the active site are often conserved across prokarytoic and eukaryotic versions, the choice of PDB structures used depends which best illustrates a conceptual point. 

    Note

    There are many ways to write abbreviated chemical equations showing NAD+/NADH and FAD/FAD2 and hydrogen ions in metabolic pathway diagrams. To make sense of them, consider a simplified mechanism for the oxidation of ethanol by alcohol dehydrogenase, as shown in the figure below. Note that there are 2 Hs on the oxidized substrate (ethanol) that are involved. One is a hydride and the other is a proton. 

     

    alcoholdehydrogenase.svg

     

    Here is a list of different and seemingly contradictory ways to write chemical equation to show changes in NAD+/NADH and H ions: 

    1. NAD+ + :H- → NADH.  This chemical equation is charge balanced and shows just the changes to the NAD+/NADH pair, but it doesn't show the proton (H+) lost from the substrate.
    2. NAD+2e- + H+ → NADH. This is the same as equation (1) but with the hydride separated into an electron pair and a protein
    3. NAD+ + H+ → NADH. This is balanced for Hs but not + charge as it really doesn't explicitly show the electron pair from the hydride added to NAD+.
    4. NAD+ → NADH + H+. This is balanced for charge but not for Hs. The extra H+ is the proton from the oxidized substrate.

    We will use example 4 above throughout this book. That equation is most useful when trying to account for the change in the number of protons in the individual reactions and in entire pathways. We will also write simplified chemical equations involving FAD (in which 2H from the substrate are added) as FAD → FADH2.

    1. Citrate Synthase (CS)

     Oxaloacetate + Acetyl-CoASH + H2O → Citrate + CoASH  ΔGo = -7.5 kcal/mol

    Acetyl CoA is a thioester. Hence it is "high energy" with respect to its hydrolysis products. The free energy released in its hydrolysis is used to drive the reaction forward.  This is important otherwise citrate would not be formed readily. This reaction feeds the end product of glycolysis into the citric acid cycle.

     

    TCA1_CitrateSynthase.svg

     

    The enzyme exists in two major conformations, an open and closed form. When the open form, which has a binding site for oxaloacetate, binds the substrate, a shift to the closed conformation forms on binding site of acetyl-CoA. These changes sequester the bound substrates and excludes water and prevents spurious hydrolysis of acetyl-CoA.The binding occurs sequentially so the kinetics following a sequential ordered mechanism. 

    Here is an animated gif that shows the conformational changes between the citrate bound version (open, green) and the citrate and CoASH bound form (blue). The image under it shows a smoother transition between the open and closed form without bound ligands (1cts, 2cts)

     

    1cts_to_2cts_Morph.gif

     

    ProteopediaIcon.pngRegulation of Citrate Synthase

    mechanism below from https://chem.libretexts.org/Bookshel...trate_Synthase

    Contributors and Attributions

    In this reaction, a C-C bond must form between the substrates.  One way to do that is to make a nucleophilic carbanion ion from the alpha carbon of acetyl CoA. Remember, this is not a decarboxylation reaction so we don't have to worry about an electron "sink" on the beta carbon. Forming the carbanion would be possible since the negative charge on the carbon can be withdrawn to the carbonyl oxygen to form an enolate.  The enolate becomes even more stable if the negative oxygen is protonated.  So this is reaction is really an aldol condensation,the addition of an enolate to an aldehyde or ketone.  The carboxylate group of an aspartic acid 375 on citrate synthase removes the acidic alpha proton on acetyl CoA, while histidine 274 donates a proton to form the neutral enol, a much more stabile molecule than the enolate anion. His 274 continues to stabilize the enol during the reaction. Bound oxaloacetate is stabilized in part by Arg 329. In the next part of the mechanism, a second histidine (320) protonates the carbonyl oxygen of oxaloacetate, activating the carbonyl carbon for nucleophilic attack by the enol in the next step to form (S)-citryl CoA. The hydrolysis of the CoASH occurs when His 320 deprotonates a water molecule, faciliating nucleophile attack on the carbonyl carbon bonded to -SCoA, forming citrate. A plausible mechanism is shown below.

     

    citratesynthaseMechfromChemLibreText.svg

    The iCn3D model below shows the structure of the complex of chicken citrate synthase (PDB ID: 6CSC) complexed with trifluoroacetonyl-CoA (a product analog) and citrate (a product). This model was chosen since both "substrates" are bound. The key residues involved in catalysis (Asp 375, His 274, Arg 329 and His 320 are shown.   Find then in the model as they interact with the products. 

    LAZY LOAD

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?8NhtVj3DJfzB1dnM6

     

     

    2. Aconitase

    Citrate ↔ Isocitrate ΔGo = +2 (rx 2a), -0.5 (rx 2b) kcal/mol; net ΔGo = + 1.5 kcal/mol

    Thinking like a chess player, who must anticipate future moves, the chemical rationale for this reaction is to move an OH to a beta position, which in a subsequent reaction is converted to a beta C=O, so it can acts as e- sink to facilitate decarboxylation in a following reaction!  The reaction is readily reversible (note the low ΔGo value) since the reactant and product are simple isomers of each other.

    TCA2_Aconitase.svg

     

    Here is a model of a S642A mutant of aconitase (1c97) bound to isocitrate. The enyzme has an inorganic Fe4S4 cluster. Each Fe in the cluster coordinates to 4 S2- iin a cubane structure, but when either citrate or isocitrate is bound, one of the Fe ion interacts with both the Os of a substate carbonxylate shown. The other two carboxylates of isocitrate are stablized through ion-ion interactions by Args 446 and 663.

    LAZY LOAD

     

     

    https://structure.ncbi.nlm.nih.gov/i...cojd8EebaGHMs5 

    Note

    Delete when ready: https://www.ncbi.nlm.nih.gov/Structu...20112&command=; pickatom 5816; select .ICT:1 or .SF4:1 | name ligands; view annotations; set annotation cdd; set view detailed view; hide annotation cdd; select .A:357 or .A:420 or .A:423 | name CoordCys; select .A:99-100 or .A:164 or .A:446 or .A:641 or .A:643 | name Cat_AA; select sets Cat_AA or CoordCys or ligands; show selection; style sidec stick; color atom; add residue number labels; set background white|||{"factor":"1.038","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"-0.3900","_y":"0.2828","_z":"0.4072","_w":"0.7760"}}

     

    The figure below shows a  plausible partial mechanism. An active site deprotonated serine abstract a proton at the S carbon. This is followed by the formation of the C-C double bond and a release of the resulting cis-aconitate from one bond to the FeS cluster. 

     

    AconitaseMechanismIsocitBound.svg

     

     

    This cis-aconitate intermediate in the interconversion of citrate and isocitrate must do a 1800 flip around the C=C double bond. This is followed by rehydration to form the other isomer The deprotonated His 101 abstracts a H from a water-bound to the FeS cluster, with the hyroxide acting as a nucleophile, which along with the redonation of a hydrogen ion on the protonated Ser 642 the alpha-carbon to complete the rehydration step in the formation of the other isomer.  

    Exercise \(\PageIndex{1}\)

    Why was the S642A mutant used to produce the structure shown in the above iCn3D model?

    Answer

    It allows the binding of substrate/product, in this case isocitrate, to an inactive enzyme as the active site serine was mutated to a non-nucleophilic alanine of similar size. Hence not bond making/breaking occurs in the complex. 

    References:

    https://collab.its.virginia.edu/acce...-_-/index.html

    https://www.ebi.ac.uk/thornton-srv/m-csa/entry/552/ 

     

    3. Isocitrate Dehydrogenase (IDH)

    Isocitrate + NAD+ → α-ketoglutarate + NADH + H+  ΔGo = -2.0 kcal/mol

    The chemical rationale should be clear.  In this step, through an oxidative decarboxyylation, the COis removed as NADH is produced.  The NADP will be reoxidized back to NAD+ in the electron transport chain, leading to ATP production (see next section). 

    TCA3_IDH.svg

     

     

    Exercise \(\PageIndex{1}\)

    Why must the oxidation reactionn precede the decarboxylation reaction?.

    Answer

    First a beta-ketoacid intermediate must form, which allows easy decarboxylation of the intermediate as the beta carbonyl provides an electron "sink" to faciliate the decarboxylation.

    There are two forms of these enzyme (IDH), a cytoplasmic (NADP+) form and a mitochondrial (NAD+ ) form. The cytoplasmic forms from various organisms are homodimer and have a common mechanism of catalysis. In contrast yeast mitochondrial IDH has two subunits, IDH1 (regulatory, binds the allosteric activator citrate and AMP) and IDH2 (catalytic, binds isocitrate and NAD+). Mammalian IDHs are tetramers of two heterodimer (αβ + αγ), which can also form a heterooctamer (αβ + αγ)2.  The alpha chain is the catalytic subunit.

    Mammalian NAD-IDHs are even more complex than yeast NAD-IDH. These enzymes are composed of three types of subunits in the ratio of 2α:1β:1γ27, which share about 40–52% sequence identity. The α and β subunits form one heterodimer (αβ) and the α and γ subunits form another heterodimer (αγ), which are assembled into a heterotetramer (α2βγ) and further into a heterooctamer (the heterotetramer and heterooctamer are sometimes called holoenzyme. The αγ heterodimer is regulated by citrate and ADP. On citrate binding to the allosteric site, a conformation change occurs to enhance isocitrate binding. ADP enhances the binding of the allosteric regulator citrate. (https://www.nature.com/articles/srep40921

    Here is a link to an iCn3D model which shows the superposition of the A chains of cytoplasmic IDH (NADP, sky blue, 4L03) and mitochondrial IDH (NAD) (6KDY, salmon). Click the 3 bar menu icon on the top left, scroll down to Alternate, and toggle back and forth between the two forms.  The structures of the alpha chains, although not identical, align well.

    LAZY LOAD

     

    Here is probable mechanism for the reaction based on the conserved catalytic site shown in the model above. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706558/

     

    IDH_mechanism.svg

    The image below shows the active site of the cytoplasmic form with bound a-ketoglutarate and NADP+.  Click it to see an interactive iCn3D model.  

    Insert Pymol image of 4L03 with link to iCn3D below: 

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?eDDuT1gRp9HFvKxz7

    LAZY LOAD

     

    The image below shows the structure of the αβ heterodimer of human IDH3 (pdb 6kdy) in complex with NAD+.  Click the image to see an interactive iCn3D model.

    Insert Pymol image with link to iCn3D below: 

    https://structure.ncbi.nlm.nih.gov/i...zFwJGumpBGXtU7

    LAZY LOAD

     

    4. α-ketoglutarate dehydrogenase

    α-ketoglutarate + NAD+ + CoASH → succinyl CoA + CO2 + NADH + H+  ΔGo = -7.2 kcal/mol

    In the last reaction, α-ketoglutarate was formed.  Oh no, you might say.  It would have been nice to form a β-ketoacid, which could easily decarboxylation.  No worries though.  We spent all of section 16.1 explaining the biochemistry used to decarboxylate another α-ketoacid, pyruvate.  The same chemistry is used to accomplish the oxidative decarboxlation  of α-ketoglutarate.  Hence we won't expand on the mechanism here. 

     

    TCA4_aKGDehydro.svg

     

    5. Succinyl-CoA synthetase (SCS)

    succinyl CoA + GDP + Pi → succinate + CoASH + GTP ΔGo = -0.8 kcal/mol

    This is the first step in which the energy change in the cycle is captured specifically in the form of a high energy (with respect to its hydrolysis product) phosphoanhydride bond in the form of GTP (and ATP in some organisms).  From a chemical step, the the cleavage of the thermodynamically unstable thioester is coupled to the endergonic synthesis of GTP.  This can transfer its terminal phosphate to ADP to make ATP in reaction that has a ΔGo of about 0 kcal/mol.  

    TCA5_SuccCoASyn.svg

    Succinyl-CoA synthases have to subunits, α and β. The enzyme in E. coli is a tetramer (α2β2) with the catalysis occurring at the αβ interface.  The alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the dimer of alpha beta-dimers with CoA bound in each α subunit to a nucleotide-binding loop.

    Two histidines, His 246 and His 142 are involved in the reaction, with His 246 becoming phosphoyrlated to form an intermediate in the reaction. A mutation of His 142 to an asparagine (H142N) essentially abolishes enzyme activity.  Different SCSs have different specificity for purine nucleoside triphosphates.  Organisms, including mammals, may have two different isoforms, one that bind ADP and one that uses GTP (as shown in most diagrams of the citric acid cycle). In E. Coli, the α subunit binds CoASH and contains His 246, which gets phosphorylated. The β subunit determines the specificity for either GTP or ATP.  In E.Coli the ATP binding site (Site II "in the ATP-grasp fold") is quite distant from the CoASH site (Site II) so phospho-His 246 must move between the sites in the dimer interface. 

    The partial reactions catalyzed but the enzyme, going form succinate to succinyl-CoA are shown below, where E is free enzyme, a . indicates a noncovalent complex, and a - represents a covalent bond (after Biochemistry 2002, 41, 537-546)

    1. E + succinyl-CoA + Pi ↔ E . succinyl-PO3 + CoASH
    2. . succinyl-PO↔  E-PO3 + succinate
    3. E-PO3 + NDP ↔ E + NTP

    Here is an abbreviated mechanism that shows only the involvement of His 246.

     SuccCoASyn_Mechanism.svg

    Kinetic analysis suggest that the three substrates bind in a specific order, catalysis occurs, and then the three products leave.  This type of reaction is called an order ter ter reaction, and is shown in the figure below. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549575/

    bottom svg:  save pptx as adobe.  OPen in Adobe illustrator.  Art Board crop to size.  The select selection icon top left.  The resize easel? so same size as art board.  Then save as svg, select svg

     

    OrderedTerTer_SuccCoASynthase.svg

    The iCn3D model below shows key alpha-chain residues in the active site, including the phosphorylated His 246 and bound CoASH.  (use iCn3Dto visualized 1CQJ, the nonphosphorylated form)

    https://structure.ncbi.nlm.nih.gov/i...oeZ1cbyoGgmQv9 

    Note

    Delete when ready:  https://www.ncbi.nlm.nih.gov/Structu...mmand=pickatom 10007; select .COA:1 | name pinkCoASH; view annotations; set annotation cdd; hide annotation cdd; set view detailed view; select .A:152-155 or .A:179 or .A:207-208 or .A:246 | name pinkActSite1; select sets pinkActSite1 or pinkCoASH; style proteins stick; color atom; set background white; add residue number labels; clear all; select .A:142 | name pinkHis142; select sets pinkHis142; add residue number labels; select sets pinkActSite1 or pinkCoASH or pinkHis142; show selection; style proteins stick; color atom|||{"factor":"1.668","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"0.6911","_y":"0.6921","_z":"0.1871","_w":"0.09134"}}

     

    Note:  1CQJ is E coli, not PO4ylated  with bound CoASH

     

    The image below shows the active the structure of pig GTP-specific succinyl-CoA synthetase in complex with succinate and CoASH is shown below (uses PDB - not mmdb - 5CAE). (NOTE: another variant exists which is specific for ATP).  Click on it to see in intereactive iCn3D mdoel.

     

    Insert Pymol image of 5cae with link to iCn3D below: 

    https://structure.ncbi.nlm.nih.gov/i...uRgGQ7u845qvA9

     

    6. Succinate Dehydrogenase

    succinate + FAD ↔ fumarate + FADH2 ΔGo = 0 kcal/mol

    The enzyme is yet another step in closing the cycle to reform oxalacetate.  It is also our first encounter with FAD as an oxidizing agent.  Its reduction product, FADH2, will be reoxidized in the the electron transport chain (mitochondrial inner membrane for eukaryotes), producing energy for ATP.   Hence it can be considered a proxy ATP-generating reaction. 

    TCA6_SuccDehydro.svg

    The succinate dehydrogenase enzyme is actually part of the larger Complex II of the electron transport chain.  Complex II has many cofactors involved in its overall activity.  t also uses an iron/sulfur cluster cofactor, similar to aconitase, which also produces a C=C doubled bonded intermediate.  We will discuss it in greater detail in the chapter on electron transport.  For now, let's concentrate on this new cofactor and oxidizing agent, FAD. Many enzymes use FAD/FADH2 in redox chemistry.

    In contrast to NAD+/NADH, the FAD/FADH2 pair stays tightly bound to the enzyme and doesn't readily dissociate.  This means that after one cycle of the enzyme (after FAD is converted to FADH2), the enzyme is functionally "dead".  Another oxidizing agent must bind to the enzyme and reoxidize FADH2 back to FAD.  The dissociation constants for FAD/FADH2 and its protein binder in a flavoprotein are often in the nanomolar range. In around 10% of flavoproteins, FAD/FADH2 are actually covalently bonded to the enzyme.

    LAZY LOAD

     

    https://structure.ncbi.nlm.nih.gov/i...JFK3yRA5t6wHNA

    https://www.ncbi.nlm.nih.gov/Structu...01113&command=; pickatom 349; select .A:56 or .FAD:1 | name PINK_FAD_H56; display interaction 3d | selected non-selected | hbonds,salt bridge,pi-cation,pi-stacking | false | threshold 3.8 5 4 3.8 5 5; select .A:48 or .A:54-59 or .A:63 or .A:177-178 or .A:232 or .A:413-414 or .FAD:1 or .Misc:6 or .Misc:18 or .Misc:26 or .Misc:32 or .Misc:42 or .Misc:45 or .TEO:1 | name PINK_5a-INTERACT; select sets PINK_5a-INTERACT or PINK_FAD_H56; show selection; color atom; set background white; add residue number labels|||{"factor":"0.7368","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"0.6297","_y":"0.4028","_z":"0.1351","_w":"0.6504"}}

     

    Here is an iCn3D model showing the residues around the Flavin

    Exercise \(\PageIndex{1}\)

    Succinate dehydrogenase is irreversibly inhibited by the toxin 3-nitropropionic acid (3np) made by some plants and funqi.  Eating moldy sugar cane has led to reported deaths.

    1.  Draw the Lewis structures of succinate and 3-nitroproionic acid.  Compare them and the total number of valence electrons in each.

    2.  Here is a link to an iCn3D model showing the interaction of 3np with enzyme.  Explain the mode of action of the toxin.

    https://structure.ncbi.nlm.nih.gov/i...cja9VKU2ZMCac9

    Answer

    These molecules are structural similar and isoelectronic (same number of electrons in their Lewis structures.

    The inhibitor 3np form a covalent adduct through the guanidino group of Arg 297.  This is a key catalytic residue which act as a general base that accepts a proton from succinate in the reaction. 

    The top image below show a very general and abbreviated mechanism for the enzyme showing just the immediate reaction of succinate with FAD.  The bottom image shows the amino acids surrounding succinate in the avian (bird) version of the enzyme (pdb 1yq4)

    succinatedehydromech.svg

    Exercise \(\PageIndex{1}\)

    One of the amino acids surrounding succinate in the figure above acts as a general base and abstracts a protein from succinate as a hydride is transfered (from a plane above) to FAD.  Go to this iCn3D to view the active site bound to xxx: https://structure.ncbi.nlm.nih.gov/i...tGGCUqs1sn6Pk8.  Which amino acid is the likely general base?  (Note:  the figure below shown general protonated states of side chains and not necessarily those involved in the proton abstraction.  Go to Analysis, Distance and Distance between 2 atoms to find the likely general base.

    Answer

    Arg 297

    https://structure.ncbi.nlm.nih.gov/i...tGGCUqs1sn6Pk8 

     

    A detailed iCn3D model of this will be explored in the chapter section on mitochondrial electron transport.

     

    7. fumarase

    fumarate + H2O ↔ L-malate ΔGo = -0.9 kcal/mol

    The chemical rationale for this reaction is clear - it is the penultimate step in the resynthesis of oxaloacetate, one of the reactants that starts the cycle, allowing the cycle to continue. This reaction  Introduce O by hydration which can be oxidized in next step to produce NADH for e- transport/ATP production. 

    TCA7_Fumarse.svg

    There are Class I (dimers containing an unstable FeS  cluster, examples A and B) and Class II (tetramer, no bound iron, oxygen stable, example C) fumarases.  Humans have both cytoplasmic and mitochondrial type II fumarses, resulting from alternative transcription of the fumarase genes.  We will consider the type II, fumarase C from E. Coli in the following discussion. 

    The tetramer contains just alpha helices and random coils and has two distinct binding site.  Site A appears to be the active site and contains a buried water molecule. Site A, formed from three of the subunits, binds competitive inhibitors such as citrate and β-(trimethylsilyl)maleate, a cis substrate for fumarase and is buried.  12 Angstroms away is site B, which is found in only one of the subunits near a pi-helix (H129 through N135) and is more surface-exposed.   Each site has a histidine, but mutation of only one H188N in the A site disrupts enzyme activity.  Both sites bind multi-carboxlates.  The role of the site B is a bit unclear, but it is mostly likely an allosteric site involved in the transfer of product (malate) from the buried site to the surface for ultimate dissociation.

    The image below shown the active site of fumarase with beta-(trimethylsilyl)maleate and citrate (1fuq)

    Insert Pymol image of 1fuq with link to iCn3D below:

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?uAoHaHdCk2N1ZPm39

    Here is a link to a iCn3D showing the key residues around the citrate, a competitive inhibitor xxx in 1fuq

    iInsert Pymol image of 1fuq with link to iCn3D below:

    https://structure.ncbi.nlm.nih.gov/i...XKtskiwjm3Ryk7

    https://www.ncbi.nlm.nih.gov/Structu...&command=color chain; view annotations; set annotation cdd; hide annotation cdd; set view detailed view; select chain !B; select chain !A; select chain !B_1; select chain !A; pickatom 6928; select .CIT:1 | name pinkLigand; select zone cutoff 5 | sets selected non-selected | false; select .A:98 or .A:100 or .A:103 or .A:139-141 or .A:231 or .A_1:324 or .A_1:326 or .B_1:187-188 | name 5ASite; select .A_1:318 or .A_1:331 | name S318_E331; select sets 5ASite or S318_E331 or pinkLigand; show selection; style proteins stick; center selection; color atom; set background white; add residue number labels|||{"factor":"1.000","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"-0.3606","_y":"-0.3454","_z":"0.1275","_w":"0.8570"}}

    Here is a plausible mechanism for the trans addition of water to fumarate.

     

    fumarasemech.svg

    Proteopedia (@proteopedia) | TwitterFumarase 2

     

    ProteopediaIcon_Scale10pc.svg

    8. Malate Dehydrogenase (MDH)

    L-malate+ NAD+ ↔ oxaloacetate + NADH + H+ ΔGo = +7.1 kcal/mol

    We are finally there!  This last reaction of the citric acid cycle produces oxaloacetate, the starting reactant, so the cycle can continue.  It also produces NADH for mitochondrial e- transport/ATP production. Notice that is is thermodynamically unfavorable (in the standard state) but the reaction is pulled to citrate formation by the first and next step of the cycle, citrate synthase. 

    TCA8_MDH.svg

    Malate dehydrogenases are found in both the cytoplasm (where it is part of the asparate-malate shuttle that moves cytoplasmic malate (and through MDH indirectly NADH) into the mitochondria  and in the mitochondria (where it is part of the citric acid cycle.  There are also NAD+ and NADP+ dependent forms.  Malate can undergo two different types of oxidation reactions, one producing oxaloacete and using NAD+, and one, an oxidative decarboxylation producing pyruvate and CO2, using NADP+.  The later is sometimes called malic enzyme.  

    Humans have two forms (MDH 1 and MDH 2) which use NAD+. The enzyme is a homodimer in humans with binding sites on both. It activity is allosterically
    regulated by citrate, and it is inhibited by many things, including ATP, ADP, AMP, fumarate, citrate, aspartate and high concentrations of oxaloacetate.  
    The enyzme is similar to lactate dehydrogenase, which we encountered in the chapter of glycolysis.  Kinetic analyses show that NAD+ binds first followed by malate.

    The iCn3D model belows shows NAD+ and malate bound to human malate dehydrogenase (pdb 4wlu)

    4wlu

    LAZY LOAD

     

     

    https://structure.ncbi.nlm.nih.gov/icn3d/share.html?Caz7sLW5sdwFAFpJ7

     

    https://www.ncbi.nlm.nih.gov/Structu...20111&command=; pickatom 9306; select .B:401-402 | name ligands; display interaction 3d | ligands non-selected | hbonds,salt bridge,halogen,pi-cation,pi-stacking | false | threshold 3.8 6 4 3.8 6 5.5; show selection; style sidec stick; color atom; add residue number labels; set background white|||{"factor":"1.050","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"-0.7109","_y":"-0.5593","_z":"-0.4232","_w":"0.05275"}}

     

    Here is an abbreviated mechanism for malate dehydrogenase. The numbers refers to the E. Coli enzyme.  Note that the hydride transferred from the malate is shown in red as a deuterium (D).  It is transferred to the re face of NAD+ to form NADH.  The carbon containing the transferred deuterium in NADH is prochiral.  Think of that as that carbon being chiral if one of the 2 Hs could be arbitrary assigned a higher priority in assigning R/S isomers. D has a higher priority than H in the Cahn/Ingold designation system.  In the reverse reaction, the D atom, which is above the plane of the ring, occupies the proR position.  The proR D is transferred back in this reversible reaction. A D was used simply to indicate the stereochemistry and to assign it in NADH to the proR position. 

    malatedehydrogenaseMech.svg

    Here is an iCn3D model showing the same active site of the E. Coli enzyme.

    https://structure.ncbi.nlm.nih.gov/i...KS4Dsf431vPMCA

    Note

    Delete when ready: https://www.ncbi.nlm.nih.gov/Structu...01115&command=; pickatom 2289; select .CIT:1 or .NAD:1 | name ligands; view annotations; set annotation cdd; hide annotation cdd; set view detailed view; select .A:81 or .A:87 or .A:150 or .A:153 or .A:177 | name aaactive; select sets aaactive or ligands; show selection; style proteins stick; color atom; set background white; add residue number labels|||{"factor":"1.000","mouseChange":{"x":"0.000","y":"0.000"},"quaternion":{"_x":"-0.6982","_y":"0.08895","_z":"0.5941","_w":"-0.3895"}}

     

    Test of electrostatic surface (Delphi) - use malate dehydrogenase as example - Doesn't Load

    We have shown many rendings of the enyzmes involved in the cycle.  Yet another one is shown below.  The display surface is the electrostatic surface potential map of the enzyme.  Red shows surfaces that are more anionic to which cationic molecules would be attracted, while blue represents more cationic surfaces to which anion would be attracted.  Note that the bound NAD+, which has many oxygens which are slightly or fully negative, is bound in a blue, cationic region.

    Click on the figure below which shows the electrostatic surface potential map of the A chain of MDH bound to NAD.

     

    MDH_electrostatics.png

    https://structure.ncbi.nlm.nih.gov/i...RK3Se5YG4AgY79

     

    Summary

    Let's do some not so simple stoichiometry for the full cycle.  Here is the net reaction (assuming that the GTP produced by succinyl-CoA synthetase is equivalent to 1 ATP).

    Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O → 2CO2 + 3NADH + FADH2 + ATP + 2H+ + CoASH

    This must seem like a lot of work to produce just 1 ATP, especially since partial, anerobic oxidation of glucose in glycolysis produced in net fashion 2 ATPs.  The key, however, is to realize that 3 NADHs and 1 FAD2 are produced, which when they are reoxidized in mitochondrial electron transport/oxidative phosphorylation, will produce multitudes of ATP. 

    Obviously, as was true for glycolysis, this main energy-extracting pathway is highly regulated. We will this in the next section. 

    The image below shows some key points about each reactions.  

     

    TCA_WedgeDashView_Notes.svg

     

     

     

     

     

    • Was this article helpful?