this chapter is about the tricarboxylic acid cycle, which takes place inside the matrix of the mitochondria and takes a three carbon molecule, acetyl coenzyme A, and through a cycle of chemical changes produces 3 molecules of NADH2, one molecule of FADH2, one molecule of GTP, and two molecules of CO2. the reactions of the TCA cycle are as follows:
1. oxaloacetate and acetyl coenzyme A are condensed into citrate via citrate synthase.
2. citrate is isomerized to isocitrate via aconase.
3. isocitrate undergoes oxidative decarboxylation to alpha-ketoglutarate via isocitrate dehydrogenase. NAD is reduced to NADH and CO2 is formed.
4. alpha-ketoglutarate undergoes another oxidative decarboxylation to succinyl CoA via alpha-ketoglutarate dehydrogenase. another NAD is reduced and CO2 is formed.
5. succinyl CoA undergoes substrate level phosphorylation via succinate thiokinase, forming succinate and GTP (from GDP).
6. succinate is oxidized to fumarate via succinate dehydrogenase. FADH2 is formed.
7. a H2O molecule is added to fumarate via fumarase, forming malate.
8. malate is oxidized back to oxaloacetate via malate dehydrogenase. NADH is formed.
the next sections cover some intricacies of the cycle. for example, the only reaction that uses FAD as an electron acceptor is the oxidation of succinate to fumarate via succinate dehydrogenase. the reason for this is that FAD can accept single electrons, forming a high energy, half reduced intermediate, whereas NAD can only accept pairs of electrons. this particular oxidation reaction involves formation of a C=C double bond, which apparently involves transferring one electron at a time- thus FAD is used instead of NAD. the enzyme that catalyzes this reaction, succinate dehydrogenase, has an FAD molecule near the active site, which accepts electrons and transfers them to an adjoining Fe-S complex, which then transfers the electrons to Coenzyme Q of the electron transport chain (recall that succinate dehydrogenase is actually "complex 2" of the electron transport chain).
another detail that is looked at: CoA contains a thioester bond, which is a C-S-C bond (as opposed to a regular C-O-C ester), which yields some special properties for molecules that contain it: first off, it is a relatively high energy bond which can provide a high and negative free energy change-- in the TCA cycle, this comes into play in the oxidation of succinyl CoA to succinate via succinate thiokinase, which provides enough energy to phosphorylate a GDP to GTP. secondly, the thioester does not participate in resonance stabilization as the regular ester does, activating the adjacent carbons to react with other molecules. this is seen in the citrate synthesis reaction, where the thioester bond on acetyl CoA activates a neighboring C, which then reacts with oxaloacetate to form citrate.
yet another detail: alpha-keto glutamate is oxidized with the help of alpha-ketoglutarate dehydrogenase, which is one of a family of three enzymes called alpha-ketoacid dehydrogenase complexes. these enzyme complexes have three subunits, labeled E1 through E3. E1 contains thiamine pyrophosphate, which cleaves a CO2 off of the alpha-keto acid, which is then oxidized and joined to a CoASH molecule by the lipoate on the E2 subunit. the electrons that lipoate received are then transferred FAD on E3, which then transfers them to NAD. the net result: these dehydrogenase complexes cleave a CO2, oxidize (hence "oxidative decarboxylation"), and combine the alpha-ketoacid with an CoA. this comes into play in the TCA cycle with the oxidative decarboxylation of alpha-ketoglutarate into succinyl CoA. later on the book talks about another enzyme in this same class, pyruvate dehydrogenase, which oxidizes pyruvate into acetyl CoA in a similar fashion.
the energetic efficiency of the TCA is touched upon: comparing the actual energetic output of the TCA cycle via the intermediates NADH and FADH2 versus what the energy output would be from combusting the intermediates in a bomb calorimeter yields an energy efficiency of ~90%. the reactions that have a large and negative free energy change: the synthesis of citrate, the oxidative decarboxylation of isocitrate as well as alpha-ketoglutarate. the reactions with a positive free change (non-spontaneous) are the oxidation of malate to oxaloacetate, and the isomerization of citrate into isocitrate. for the malate reaction, this means that the equilibrium favors malate, leaving a low concentration of oxaloacetate.
the low concentration of oxaloacetate facilitates regulation of the first reaction of the TCA cycle, citrate synthesis from oxaloacetate and Acetyl CoA. the TCA cycle is regulated in many different places, generally inhibited by the products that it creates and stimulated by buildup of reactants so as to maintain homeostasis and adequate ATP synthesis. in this fashion, citrate synthase is inhibited by the product it creates, citrate and stimulated by the reactant it uses, oxaloacetate. oxaloacetate production is itself stimulated by the NAD/NADH ratio, which in turn reflects the ATP usage: demonstrating that all activity in the cycle is ultimately correlated back to ATP usage. another example of enzymatic regulation is alpha-ketoglutarate dehydrogenase being activated by ADP (reflecting the need for more oxidative activity), inhibited by NADH (reflecting the lack of oxidative activity downstream -- therefore signalling the reaction to stop), and also activated by Ca2+ (which is released intracellularly during rapid muscle contraction, reflecting the need for greater oxidative activity).
after regulation we then take a look at the synthesis and usage of the precursors and intermediates of the TCA cycle, beginning with acetyl coA and pyruvate. this is an important reaction, as it converts the product of aerobic glycolysis, pyruvate, into the first compound of the TCA cycle, acetyl CoA. as was mentioned earlier, pyruvate is converted to Acetyl CoA by one of the three enzymes in the alpha-ketoacid dehydrogenase complex family, called pyruvate dehydrogenase complex (PDC). PDC is intricately regulated both by the reactant/product system described earlier (inhibited by NADH and acetyl CoA, two products of the pyruvate oxidation reaction), as well as a system of two enzymes which deactivate/activate it by phosphorylation/removing a phosphate, named PDC kinase and PDC phosphatase. PDC kinase is inhibited by high ADP levels and high pyruvate levels- in the first case the high ATP usage signals PDC kinase to stop its inhibitory activity of PDC, allowing pyruvate to be oxidized. in the second case the high pyruvate levels signals an excess of reactant, which produces the same effect. PDC phosphatase removes the phosphate from PDC, allowing it to catalyze the oxidation of pyruvate. it is stimulated by Ca2+, which can signal rapid muscle contraction and reflect the need for greater oxidative activity- as in with alpha-ketoglutarate dehydrogenase.
the intermediates of the TCA cycle are used for other purposes, especially so in the liver, where the TCA is called an "open cycle" for this reason. oxaloacetate and alpha-ketoglutarate are both used for amino acid synthesis, citrate is used for fatty acid synthesis, succinyl CoA is used for heme synthesis, and malate is used for gluconeogenesis. additionally, in skeletal muscle, alpha-ketoglutarate is converted into glutamine, and in the brain, converted into the neurotransmitter GABA. to replenish the intermediates used in these reactions (called "efflux" of intermediates), "anaplerotic" reactions take place. the major anaplerotic reactions are: pyruvate carboxylase adding an activated CO2 to pyruvate to form and replenish oxaloacetate. glutamate being converted back into alpha-ketoglutarate via transaminases or glutamate dehydrogenases. valine or isoleucine being converted into succinyl CoA. other amino acids being converted to fumarate and oxaloacetate.
questions
1. what are the byproducts of the TCA cycle?
2. which reactions produce NADH as a byproduct?
3. which reaction produces FADH2 as a byproduct?
4. why is FAD used as an electron acceptor in the oxidation of succinate as opposed to NADH?
5. describe the role of succinate dehydrogenase in the oxidation of succinate.
6. what is the relationship of the NAD/NADH ratio to oxidative enzymes?
7. what is unique about the thioester bond in CoA and how is it used in the TCA cycle?
8. what are the three components to alpha-ketoacid dehydrogenase complexes?
9. what is the energetic efficiency of the TCA cycle?
10. which reactions of the TCA cycle have a large and negative change in free energy?
11. which reactions of the TCA cycle have a positive change in free energy?
12. describe the regulation of the first step in the TCA cycle.
13. describe the regulation of isocitrate dehydrogenase.
14. describe the regulation of alpha-ketoglutarate dehydrogenase.
15. what does the pyruvate dehydrogenase complex do?
16. describe the actions of PDC kinase on PDC and what it is influenced by.
17. describe the actions of PDC phosphatase on PDC and what it is influenced by.
18. why is the TCA cycle in the liver called an "open cycle"?
19. what are examples of efflux of TCA intermediates in the brain and skeletal muscle?
20. what are anaplerotic reactions?
21. describe the actions of the pyruvate carboxylase enzyme. what is it activated by?
22. what are the other major anaplerotic reactions that replenish TCA intermediates?
answers
1. 3 molecules of NADH, 1 molecule of FADH2, 2 molecules of CO2.
2. oxidative decarboxylation of isocitrate to alpha-ketoglutarate, oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA, oxidation of malate to oxaloacetate.
3. oxidation of succinate to fumarate via succinate dehydrogenase.
4. FAD can accept single electrons, forming a half-reduced intermediate. the oxidation of succinate involves the formation of a double bond, which involves transfer of single electrons, making FAD better suited as an electron acceptor.
5. succinate dehydrogenase contains a covalently bound FAD molecule near its active site, which can accept electrons and transfer them directly to an Fe-S complex, which then donates them directly to Coenzyme Q (of the electron transport chain)
6. free NAD+ molecules bind to dehydrogenases and are reduced to NADH, which can then bind to and inhibit the oxidation activity of other dehydrogenases. thus the NAD/NADH regulates the activity of oxidative enzymes to coordinate the rate of fuel oxidation with ATP production.
7. the thioester bond is unique because it is a high energy bond (-13kJ/mol), and it differs from regular oxygen esters in that it does not share its electrons, and therefore does not allow for resonance stabilization, thereby allowing neighboring carbons to be activated for different reactions. this is demonstrated in citrate's synthesis, where the alpha-carbon in CoA is activated by the thioester, allowing it to undergo a condensation reaction with oxaloacetate. the release of energy from the breakage of the thioester bond in CoA is harnessed in the reaction of succinyl CoA to succinate, where an enzyme-bound phosphate is transferred to GDP to create GTP.
8. TPP's, lipoases, and FAD. thiamine pyrophosphates cleave the C-C bonds next to the carboxyl group, which is how the CO2 is freed in the decarboxylation reactions. lipoases are responsible for transferring the acyl portion of the alpha-ketoacid to CoASH, as well as accepting and donating electrons to FAD, which then transfers them to NAD.
9. ~90%
10. synthesis of citrate, oxidation of isocitrate and alphaketoglutarate.
11. oxidation of malate, isomerization of citrate.
12. the synthesis of citrate from oxaloacetate and acetyl coA is regulated by the concentration of oxaloacetate, (availability of reactant), as well as concentration of citrate (which can competitively inhibit the reaction). the concentration of oxaloacetate is regulated by the NAD/NADH ratio, which is a reflection of the level of oxidative phosphorylation, and the concentration of citrate is regulated by the level of activity of the isocitrate dehydrogenase enzyme.
13. the presence of ADP causes isocitrate dehydrogenase to activate all subunits on the enzyme, allowing the reaction to proceed more rapidly (lowering the michaelis constant). NADH inhibits isocitrate activity.
14. this enzyme is inhibited by NADH, activated by Ca2+ and ADP.
15. the pyruvate dehydrogenase complex (PDC) is an enzyme that catalyzes the oxidation of pyruvate into acetyl coA, connecting glycolysis with the TCA cycle.
16. PDC kinase phosphorylates and deactivates PDC. PDC kinase is inhibited by ADP, pyruvate and activated by Acetyl CoA and NADH: when ATP is used up, the ADP concentration rises, inhibiting PDC kinase activity, leaving PDC in the active form, allowing it to oxidize pyruvate to acetyl CoA to be used in the TCA cycle and ultimately ATP production. (thus high ADP levels stimulate more ATP production). presence of pyruvate has the same inhibitory effect on PDC kinase, allowing PDC to oxidize pyruvate. (thus high pyruvate levels allow PDC to oxidize pyruvate). Acetyl CoA and NADH are products of the PDC reaction which activates PDC kinase, which phosphorylates PDC and inhibits its activity (thus accumulation of the products from the oxidation of pyruvate inhibits the oxidation of pyruvate)
17. PDC phosphatase, the enzyme that activates PDC, is activated by Ca2+ -- as in when Ca2+ is released from sarcoplasmic reticulum during smooth muscle contraction (thus rapid muscle contraction stimulates pyruvate oxiation).
18. because of the high efflux of intermediates that are used for amino acid synthesis (oxaloacetate, alpha-ketoglutarate), fatty acid synthesis (citrate), heme synthesis (succinyl CoA), and gluconeogenesis (malate).
19. in the brain, alpha-ketoglutarate is used to form GABA, a neurotransmitter. in skeletal muscle, it is converted to glutamine, which is transported in the blood to other tissues.
20. reactions that replenish the intermediates of the TCA cycle via carbohydrates or amino acids.
21. pyruvate carboxylase is an enzyme that catalyzes the anaplerotic reaction which replenishes oxaloacetate. it does so by addition of an activated CO2 to pyruvate to form oxaloacetate, expending one ATP in the process. it is activated by Acetyl CoA: if oxaloacetate is used up for other reactions such as amino acid synthesis, then the citrate synthesis from oxaloacetate + acetyl CoA will not occur, and acetyl CoA will accumulate. the buildup of acetyl CoA will then activate pyruvate carboxylase to replenish oxaloacetate so that the TCA cycle can get started. (thus higher Acetyl CoA stimulates the replenishing of oxaloacetate)
22. glutamate replenishes alpha-ketoglutarate by transamination or glutamate dehydrogenase activity. carbon skeletons of caline and isoleucine replenish succinyl CoA. other amino acids are degraded to fumarate and oxaloacetate, mainly in the liver.
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