this chapter looked at the synthesis, processing, and packaging of fatty acids in the liver and adipose tissue. the first section looked at where the precursors for fatty acid synthesis come from: acetyl CoA is required for fatty acid synthesis and is created in the mitochondria from pyruvate via pyruvate dehydrogenase, then combined with oxaloacetate to form citrate. citrate is shuttled out into the cytosol and cleaved back into acetyl CoA and oxaloacetate (which is then recycled back to pyruvate).
the synthesis of fatty acids begins with the conversion of acetyl CoA to malonyl CoA via the enzyme acetyl CoA carboxylase. (recall from chapter 23 that malonyl CoA is an inhibitor of beta oxidation of fatty acids). this is the rate limiting step of fatty acid synthesis and is regulated by a number of different factors. first, it is inhibited by the products of fatty acid synthesis, malonyl CoA, palmitate, and palmitoyl CoA, and also stimulated by a buildup of reactants- acetyl CoA, citrate, glucose. secondly, acetyl CoA carboxylase is inhibited by a high insulin level, which activates a phosphatase that inactivates the enzyme, while stimulated by high AMP levels (signaling the need for more energy), which activates a protein kinase that activates the enzyme.
the steps for the synthesis of fatty acids:
1. an acetyl moeity attaches to the sulfhydryl group (short arm) of the fatty acid synthase complex and then is transferred to the cysteine-sulfhydryl group (long arm).
2. a malonyl moeity attaches to the short arm and undergoes a condensation reaction with the acetyl CoA from step 1.
3. the keto-acyl that is formed in step two is reduced to an alcohol, forms a double bond via removal of water, and reduced again to remove the double bond. the net result of steps 2 and 3 is the addition of 2 carbons to the ∆-end of the fatty acid chain.
4. the keto-acyl chain is transferred back to the long arm, and another malonyl moeity is attached to the short arm.
5. the keto-acyl chain and malonyl combine via a condensation reaction and the elongation process continues.
6. when the fatty acyl chain reaches 16 carbons, hydrolysis occurs and palmitate is released.
palmitate can then be elongated and desaturated. elongation occurs in a process similar to fatty acid synthesis, except the fatty acyl chain attaches to coenzyme A rather than the ACP sulfhydryl group. a common elongation reaction is that of palmityl CoA to stearyl CoA (C18). desaturation occurs in the endoplasmic reticulum and requires molecular oxygen, NADH, and cytochrome b5. in humans, this process can only occur up to carbon ∆9; thus carbons ∆10 through ∆16 on the palmitate produced by fatty acid synthesis can not be de-saturated. omega 3 and 6 fatty acids fit into this category (3 and 6 carbons from the opposite end correspond to carbons ∆14 and ∆11) and thus must be obtained from plant oils and fish oils. plant oils contain linoleic acid and alpha-linolenic acid, both of which are used as precursors for eicosanoids synthesis.
the next section talks about what happens to fatty acids after they are synthesized in the liver. fatty acids are then combined into triacylglycerols- the glycerol backbone is created either through phosphorylation of glycerol via glycerol kinase, or through the glycolytic intermediate G3P. the process of forming a triacylglycerol is as follows: two fatty acyl CoA's are combined with a glycerol 3-phosphate molecule to form phosphatidic acid. phosphatidic acid is then dephosphorylated to form a diacylglycerol, to which another fatty acyl CoA is added, forming a triacylglycerol. the TG's are then packaged into very-low-density-lipoproteins (VLDL), which are similar to chylomicrons (recall from lipid digestion) in that they are hydrophilic droplets containing TG's, cholesterol, and apoproteins. The nascent VLDL is released into the bloodstream, where it matures upon receiving apoproteins CII and E from a HDL molecule.
the fate of VLDL's is discussed: in circulation, VLDL encounters the enzyme lipoprotein lipase (LPL), located on the endothelium basement membrane, which cleaves the TG's in the VLDL's into glycerol and fatty acids. the LPL in skeletal muscle has a high affinity for the TG's, allowing skeletal muscle to metabolize fatty acids even if the concentration is low. in contrast, the LPL in adipose tissue has a lower affinity for the TG's, and is consequently activated during the fed state when the concentration of VLDL's in circulation is high. once the TG's have been removed from the VLDL, it becomes an intermediate or low density lipoprotein. in adipose tissue, the glycerol backbone is released back into the bloodstream and back to the liver to be recycled, since adipose tissue does not have the glycerol kinase necessary to reuse glycerol for TG synthesis. during the fed state, the high insulin/glucagon ratio stimulates synthesis of LPL, ushering fatty acids into the adipose cells, where they are repackaged into TG's in a similar fashion to what was described above. in the fasting state, a glucagon sensitive lipase cleaves the stored TG's into fatty acids, which are then released into circulation, where they can be metabolized for energy (see chapter 23 notes).
questions
introduction...
1. when are fatty acids synthesized?
2. where does fatty acid synthesis take place?
3. where is the fatty acid synthase complex located?
4. what are the two possible fates for pyruvate in the mitochondria?
5. describe the production of acetyl coA for fatty acid synthesis.
6. why does acetyl CoA need to be produced in the mitochondria? why is it converted into citrate?
7. where does the NADPH required for fatty acid synthesis come from?
8. describe the recycling of oxaloacetate back to pyruvate in the cytosol.
9. acetyl coa synthesis in the mitochondria is stimulated by...
10. why doesn't citrate just get used in the TCA cycle after being synthesized in the mitochondria?
fatty acid synthesis...
11. describe the synthesis of malonyl CoA.
12. what is the rate limiting enzyme of fatty acid synthesis?
13. what is acetyl coA carboxylase regulated by?
14. describe the first steps of fatty acid synthesis on the fatty acid synthase enzyme.
15. describe how the fatty acid chain is elongated.
16. at what point is the fatty acid released from fatty acid synthase?
17. where does the elongation of palmitate occur?
18. what is the main difference between the processes of fatty acid synthesis and fatty acid elongation?
19. what is the main elongation reaction that occurs in the body?
desaturation...
20. what does desaturation of fatty acids require?
21. what are the most common desaturation reactions that take place in the body?
22. what is the limitation in the body's ability to unsaturate fatty acids?
23. where do we obtain w6 and w3 fatty acids?
24. what are linoleic and alpha-linolenic converted to in the body?
25. where do the w3 and w6 fatty acids in fish oil come from?
packaging...
26. where does G3P come from in the liver?
27. where does G3P come from in adipose tissue?
28. describe the synthesis of a triacylglycerol.
29. what is glyceroneogenesis? where and when does it occur? what is it regulated by?
30. what is a VLDL?
31. what is the major apoprotein in VLDL's?
32. how do VLDL's differ from chylomicrons?
33. how do VLDL's become mature?
fate...
34. what is the fate of VLDL's?
35. what is LPL activated by?
36. describe the difference in Km of LPL between muscle and adipose tissue.
37. what happens to the VLDL after the TG's have been removed?
38. describe what happens to fatty acids in adipose tissue in the fed state.
39. what happens to glycerol in the adipose tissue?
40. describe what happens to TG's in adipose tissue in the fasting state.
answers
1. whenever an excess of calories is consumed.
2. mostly in the liver, also in the adipose tissue.
3. in the cytosol
4. conversion to oxaloacetate via pyruvate carboxylase or acetyl coA via pyruvate dehydrogenase.
5. pyruvate in the mitochondria is converted to acetyl coA and combined with oxaloacetate to form citrate. citrate is shuttled out into the cytosol to and split back into oxaloacetate and acetyl coA via citrate lyase.
6. because pyruvate dehydrogenase is only found in the mitochondria, and acetyl coA can not directly cross the mitochondrial membrane.
7. from the pentose phosphate pathway and also from the recycling of oxaloacetate back to pyruvate in the cytosol.
8. oxaloacetate is reduced by malate dehydrogenase into malate. malate is oxidatively decarboxylated into pyruvate via malic enyzme. NADPH is formed in the second step.
9. a high insulin/glucagon ratio.
10. because of allosteric inhibition of isocitrate dehydrogenase.
11. acetyl CoA is oxidatively decarboxylated to malonyl CoA via acetyl CoA carboxylase.
12. acetyl CoA carboxylase.
13. acetyl CoA carboxylase is active in the dephosphorylated form; when insulin levels are high, it is dephosphorylated by a phosphatase. when energy supplies are low, an AMP-dependent protein kinase phosphorylates it back into the inactive form. also, malonyl CoA and palmitoyl CoA (an intermediate and a product of fatty acid synthesis) inhibit while acetyl CoA and citrate (reactants) stimulate the enzyme.
14. an acetyl CoA moiety is transferred to the cysteine-sulfhydryl group, then transferred to the sulfhydryl group. malonyl coA attaches to the sulfhydryl group and then undergoes a condensation reaction with the acetyl, forming a 4 carbon keto-acyl chain and releasing CO2.
15. the carboxyl at the omega end of the four carbon keto-acyl chain is then reduced to an alcohol, has water removed to form a double bond, and reduced again to form a single bond. the keto-acyl chain is then transferred to the cysteine-sulfhydryl group and malonyl is added to it in the same fashion as the first step.
16. when the fatty acid is 16 carbons long (palmitate), it is released via hydrolysis.
17. in the endoplasmic reticulum.
18. in fatty acid elongation, the fatty acyl attaches to a CoA on the fatty acid synthase complex, as opposed to a phosphopantetheinyl group.
19. elongation of palmitate (C16) to stearate (C18).
20. molecular oxygen, NADH, and cytochrome b5.
21. introduction of a double bond in position ∆9 as in the conversion of palmitic acid to palmitoleic acid and stearic acid to oleic acid.
22. the body can only introduce a double bond up to carbon ∆9. thus it can not produce omega-3 or 6 unsaturated fatty acids, which involve carbons beyond that point.
23. mainly from dietary plant oils: linoleic acid (18:2, ∆9,12), alpha-linolenic acid (18:3, ∆9,12,15)
24. arachidonic acid, eicosapentaenoic acid, precursors for eicosanoids.
25. the phytoplankton that they consume.
26. phosphorylation of glycerol via glycerol kinase, or from reduction of DHAP from glycolysis.
27. only from glucose via reduction of DHAP (there is no glycerol kinase enzyme in adipose)
28. two fatty acids combine with the glycerol 3-P to form phosphatidic acid, which is then phosphorylated to form diacylglycerol. a third fatty acid is added to diacylglycerol to form a triacylglycerol.
29. glyceroneogenesis is the formation of new glycerol molecules from gluconeogenic precursors such as alanine, aspartate, and malate. it occurs in the adipose tissue during the fasting state and is regulated by the presence of PEPCK enzyme.
30. a very low density lipoprotein particle which is packaged in the golgi apparatus, contains TG's, cholesterol, phospholipids, proteins, and released by the liver into circulation.
31. apoB-100, related to the apoB-48 in chylomicrons.
32. they are more dense because they contain a smaller proportion of TG's.
33. upon acquisition of apoproteins CII and E from HDL particles in circulation.
34. the TG's in the VLDL's get cleaved by lipoprotein lipase present in the endothelium basement membrane.
35. the C-II apoprotein.
36. muscle tissue LPL has a low Km, allowing muscle to use fatty acids even with concentration of VLDL's are low. adipose tissue LPL has a high Km and is most active in the fed state, when the concentration of circulating fatty acids is high.
37. they form intermediate-density lipoproteins or low density lipoproteins.
38. during the fed state, high insulin levels stimulate synthesis of LPL in adipose tissue capillaries, which releases fatty acids from VLDL's (and chylomicrons). fatty acids are activated and form triacylglycerols using the same pathway as in the liver.
39. since adipose tissue has no glycerol kinase, it can't use glycerol to produce more TG's. thus glycerol travels back into circulation to the liver.
40. glucagon activates a hormone sensitive lipase, which cleaves fatty acids off of TG's, which are released into the bloodstream along with glycerol.
real questions
where do fatty acid elongation and desaturation occur in the body?
why can't double bonds be produced beyond carbon 9 during unsaturation of fatty acids?
Showing posts with label pyruvate. Show all posts
Showing posts with label pyruvate. Show all posts
Thursday, January 22, 2009
Tuesday, November 25, 2008
biochem: mark's medical biochem chapter 20- TCA cycle
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.
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.
Thursday, November 20, 2008
biochem: mark's medical biochem chapter 22- glycolysis
this chapter introduces glycolysis and its role in cellular energy production. glycolysis is basically the first step in breaking down glucose for use in energy production. in aerobic glycolysis, one molecule of glucose is broken down into two molecules of pyruvate, in the process forming 2 molecules of NADH and 2 molecules of ATP. in anaerobic glycolysis, one molecules of glucose produces lactate and 2 molecules of ATP. the reactions for aerobic glycolysis are as follows:
1. glucose is phophorylated to glucose-6-phosphate via hexokinase.
2. glucose 6-phophate is isomerized to fructose 6-phosphate via phosphoglucose isomerase.
3. fructose 6-phosphate is phosphorylated to fructose 1,6 bisphosphate via phosphofructokinase-1.
4. fructose 1,6 bisphosphate is cleaved into two triose phosphates via an aldolase.
5. the triose phophates isomerize into glyceraldehyde 3-phosphate (G3P).
6. G3P is oxidized by G3P dehydrogenase, which reduces NAD+ to NADH2 and transfers a high energy acyl phosphate bond onto G3P, forming 1,3 bisphophoglycerate.
7. 1,3 bisphosphoglycerate phosphorylates ADP via phosphoglycerate kinase and becomes 3 phosphoglycerate.
8. 3-phosphoglycerate is isomerized to 2-phosphoglycerate via phosphoglyceromutase.
9. 2-phosphoglycerate loses an H2O and becomes phosphoenolpyruvate (PEP) via enolase.
10. PEP phosphorylates another ADP via pyruvate kinase and becomes pyruvate.
reactions 1 through 4 are known as the preparative phase, in which glucose is basically phosphorylated twice and cleaved into two 3 carbon molecules. reactions 5-10 are known as the ATP generating phase, because the phosphate groups from the 3 carbon molecules are then transferred onto ADP. after the first phosphate group is transferred in reaction 7, reactions 8 and 9 basically transform the remaining phosphate group from a low energy to a high energy one so that it can also be transferred to ADP.
in reaction 6, NAD+ is used as an electron acceptor during the oxidation of G3P. thus, the NADH2 must be constantly reoxidized to NAD+ in order to maintain a source for transferring electrons. in aerobic glycolysis, this problem is solved by shuttling NADH2's electrons across the mitochondrial membrane (shuttling is needed because the membrane is impermeable to NAD), where they can be used to power oxidative phosphorylation. the most common shuttle is the G3P shuttle: NADH2 reduces DHAP to G3P via G3P dehydrogenase. G3P then diffuses across the mitochondrial membrane and transfers the electrons to FAD, forming FADH2, which can then donate its electrons to coenzyme Q in the electron transport chain. the other shuttle mechanism is the malate aspartate shuttle, in which NADH2 reduces oxaloacetate to malate, which is then shuttled across the mitochondrial membrane via a translocase, and then donates its electrons to mitochondrial NAD+, reforming oxaloacetate. oxaloacetate is then transaminated to aspartate, which can then be shuttled back out the mitochondria via another translocase, and once it is back, transaminated back into oxaloacetate and ready to accept electrons from NADH2 again.
the net ATP production from the G3P shuttle is 3 moles of ATP per mole of glucose, whereas the malate aspartate shuttle produces 5 moles of ATP. combining this with the ATP produced in the ATP-generating phase of glycolysis (2 ATP per glucose), and taking into account the pyruvate, which can be further oxidized in the mitochondria to produce an additional 12.5 ATP (25 ATP per glucose): this means in aerobic glycolysis, total ATP production for each molecule of glucose is either 3+2+25=30 when the G3P is in use or 5+2+25=32 when the malate aspartate shuttle is in use.
when oxygen is not readily available or mitochondria are not present (as in red blood cells, where oxidative metabolism might interfere with hemoglobin's interaction with oxygen, or in the eye, where mitochondria would deflect incoming light), or if cellular demands for energy exceeds that which is produced via aerobic processes, then anaerobic glycolysis is used. in order to regenerate the NAD+ to serve as the electron acceptor in reaction 6, NADH2 reduces pyruvate into lactic acid, which dissociates into lactate and proton (which has the potential to decease body pH). pyruvate therefore cannot be further oxidized and therefore the energy production in anaerobic glycolysis is quite lower, requiring tissues to work 15 times as fast to produce the same energy output. whereas in aerobic glycolysis the total energy production can be up to 32 moles of ATP per mole of glucose, in anaerobic glycolysis, the total energy production is only 2 moles of ATP (from the ATP-generating phase of glycolysis)
questions
1. describe the process of glycolysis and what it produces.
2. what are the two phases of glycolysis and what happens in each?
3. what is the first step in glycolysis and what does it produce?
4. what are the next steps in the preparative phase of glycolysis?
5. describe the oxidation of G3P.
6. what is substrate level phosphorylation?
7. where is the first phosphorylation of ADP?
8. how is the low energy phosphate bond in 3 phosphoglycerate transformed into a high energy phosphate bond that can be transferred to ADP?
9. what is the final reaction of glycolysis?
10. what is the total free energy change from glycolysis?
11. why does NADH need to be reoxidized and what are the two pathways in which this is accomplished?
12. what are the two fates for pyruvate?
13. why are reducing equivalent shuttles required?
14. describe the G3P shuttle.
15. describe the malate-aspartate shuttle.
16. describe anaerobic glycolysis.
17. what is the ATP production of the G3P vs. the malate-aspartate shuttle?
18. describe the difference of ATP production possible in aerobic vs. anaerobic glycolysis.
19. how is anaerobic glycolysis related to body pH?
20. when do cells or tissues use anaerobic glycolysis vs. aerobic?
answers
1. glycolysis is the splitting of glucose into pyruvate and production of 4 ATP molecules per molecule of glucose.
2. the preparative phase, in which glucose is phosphorylated twice by ATP and cleaved into two triose phosphates, and the ATP-generating phase, in which the triose phosphates produce 2 ATP each.
3. phosphorylation of glucose yields glucose-6-phosphate via hexokinases.
4. isomerization of glucose 6-phosphate via phosphoglucose isomerase to fructose 6-phosphate, phosphorylation to fructose 1,6 bisphosphate via phosphofructokinase-1 (PFK-1), and cleavage into two triose molecules (glyceraldehyde 3-phosphate) via aldolase.
5. G3P is oxidized by glyceraldehyde-3-phosphate dehydrogenase, which has a cysteine residue near the active site which accepts a high energy acyl phosphate and transfers it to G3P. in this process, NADH and a high energy phosphate bond are formed.
6. the formation of a high energy phosphate bond without oxygen via direct transfer of a phosphate bond from a high energy intermediate.
7. 1,3 bisphosphoglycerate transfers its phosphate group to ADP via phosphoglycerate kinase, forming 3 phosphoglycerate.
8. the low energy phosphate bond in 3-phosphoglycerate is first shifted to the middle carbon via phosphoglyceromutase, and then transformed into a higher energy phosphate bond via enolase, which removes a water molecule and turns the compound into phosphoenolasepyruvate.
9. transfer of the high energy phosphate bond phospho enol pyruvate to ADP, turning the compound into pyruvate.
10. -22kcal
11. NADH needs to be reoxidized in order to continually accept electrons during the oxidation of G3P via G3P dehydrogenase. when oxygen is available, reducing equivalents are shuttled across the mitochondrial membrane to the electron chain. when oxygen is not available, NADH transfers its electrons to pyruvate, which is then reduced to lactate.
12. pyruvate can be reduced to lactate in anaerobic glycolysis, or can be converted into acetyl coenzyme A and further oxidized in aerobic glycolysis.
13. because the mitochondrial membrane is impermeable to NADH.
14. the G3P shuttle is the main shuttle mechanism in which cytosolic NAD+ is regenerated by G3P dehydrogenase, which transfers the electrons from NADH2 to DHAP, forming G3P. G3P then diffuses across the mitochondrial membrane and donates its electrons to FAD, forming FADH2 and reforming DHAP, which can then diffuse back and be reused in the reaction.
15. the malate-aspartate shuttle is the other way that NAD+ is regenerated: NADH transfers its electrons to oxaloacetate, reducing it to malate, which is then transported across the mitochondrial membrane via a translocase. in the membrane, the malate reduces NAD to NADH2, which can then be used in the electron transport chain. the oxaloacetate that is formed is transaminated to aspartate, which is transported back across the mitochondrial membrane via another translocase, and transaminated back into oxaloacetate in the cytosol.
16. anaerobic glycolysis occurs when the the oxidative capacity of a cell is limited. in this reaction, the electrons from NADH2 are donated to pyruvate, creating lactic acid, catalyzed by the enzyme lactate dehydrogenase.
17. the G3P shuttle produces 1.5 moles of ATP per NADH2, whereas the malate-asparate shuttle produces 2.5 moles of ATP per NADH2.
18. in anaerobic glycolysis, pyruvate is reduced to lactate and thus cannot produce any more ATP besides the two moles (per glucose) that is produced during glycolysis. in aerobic glycolysis, in addition to these two ATP's, the pyruvate is oxidized and used in the mitochondria to produce 25 ATP (per glucose), and the NADH produced can add another 3 (if using the G3P pathway) or 5 (if using the malate aspartate pathway) ATP molecules.
19. lactic acid is produced from the reduction of pyruvate in anaerobic glycolysis via lactate dehydrogenase. at the normal intracellular pH of 7.35, lactic acid dissociates into lactate and H+, which is then transferred out of the cell and eventually into the blood, where it can influence body pH.
20. tissues that have a low ATP demand and low O2 availability. also, blood cells have undergo anaerobic glycolysis because aerobic metabolism might interfere with the loading and unloading of O2 from hemoglobin. also, in other tissues with mitochondria and O2 availability, anaerobic glycolysis is used when the cell's energetic demands are not met by aerobic processes.
1. glucose is phophorylated to glucose-6-phosphate via hexokinase.
2. glucose 6-phophate is isomerized to fructose 6-phosphate via phosphoglucose isomerase.
3. fructose 6-phosphate is phosphorylated to fructose 1,6 bisphosphate via phosphofructokinase-1.
4. fructose 1,6 bisphosphate is cleaved into two triose phosphates via an aldolase.
5. the triose phophates isomerize into glyceraldehyde 3-phosphate (G3P).
6. G3P is oxidized by G3P dehydrogenase, which reduces NAD+ to NADH2 and transfers a high energy acyl phosphate bond onto G3P, forming 1,3 bisphophoglycerate.
7. 1,3 bisphosphoglycerate phosphorylates ADP via phosphoglycerate kinase and becomes 3 phosphoglycerate.
8. 3-phosphoglycerate is isomerized to 2-phosphoglycerate via phosphoglyceromutase.
9. 2-phosphoglycerate loses an H2O and becomes phosphoenolpyruvate (PEP) via enolase.
10. PEP phosphorylates another ADP via pyruvate kinase and becomes pyruvate.
reactions 1 through 4 are known as the preparative phase, in which glucose is basically phosphorylated twice and cleaved into two 3 carbon molecules. reactions 5-10 are known as the ATP generating phase, because the phosphate groups from the 3 carbon molecules are then transferred onto ADP. after the first phosphate group is transferred in reaction 7, reactions 8 and 9 basically transform the remaining phosphate group from a low energy to a high energy one so that it can also be transferred to ADP.
in reaction 6, NAD+ is used as an electron acceptor during the oxidation of G3P. thus, the NADH2 must be constantly reoxidized to NAD+ in order to maintain a source for transferring electrons. in aerobic glycolysis, this problem is solved by shuttling NADH2's electrons across the mitochondrial membrane (shuttling is needed because the membrane is impermeable to NAD), where they can be used to power oxidative phosphorylation. the most common shuttle is the G3P shuttle: NADH2 reduces DHAP to G3P via G3P dehydrogenase. G3P then diffuses across the mitochondrial membrane and transfers the electrons to FAD, forming FADH2, which can then donate its electrons to coenzyme Q in the electron transport chain. the other shuttle mechanism is the malate aspartate shuttle, in which NADH2 reduces oxaloacetate to malate, which is then shuttled across the mitochondrial membrane via a translocase, and then donates its electrons to mitochondrial NAD+, reforming oxaloacetate. oxaloacetate is then transaminated to aspartate, which can then be shuttled back out the mitochondria via another translocase, and once it is back, transaminated back into oxaloacetate and ready to accept electrons from NADH2 again.
the net ATP production from the G3P shuttle is 3 moles of ATP per mole of glucose, whereas the malate aspartate shuttle produces 5 moles of ATP. combining this with the ATP produced in the ATP-generating phase of glycolysis (2 ATP per glucose), and taking into account the pyruvate, which can be further oxidized in the mitochondria to produce an additional 12.5 ATP (25 ATP per glucose): this means in aerobic glycolysis, total ATP production for each molecule of glucose is either 3+2+25=30 when the G3P is in use or 5+2+25=32 when the malate aspartate shuttle is in use.
when oxygen is not readily available or mitochondria are not present (as in red blood cells, where oxidative metabolism might interfere with hemoglobin's interaction with oxygen, or in the eye, where mitochondria would deflect incoming light), or if cellular demands for energy exceeds that which is produced via aerobic processes, then anaerobic glycolysis is used. in order to regenerate the NAD+ to serve as the electron acceptor in reaction 6, NADH2 reduces pyruvate into lactic acid, which dissociates into lactate and proton (which has the potential to decease body pH). pyruvate therefore cannot be further oxidized and therefore the energy production in anaerobic glycolysis is quite lower, requiring tissues to work 15 times as fast to produce the same energy output. whereas in aerobic glycolysis the total energy production can be up to 32 moles of ATP per mole of glucose, in anaerobic glycolysis, the total energy production is only 2 moles of ATP (from the ATP-generating phase of glycolysis)
questions
1. describe the process of glycolysis and what it produces.
2. what are the two phases of glycolysis and what happens in each?
3. what is the first step in glycolysis and what does it produce?
4. what are the next steps in the preparative phase of glycolysis?
5. describe the oxidation of G3P.
6. what is substrate level phosphorylation?
7. where is the first phosphorylation of ADP?
8. how is the low energy phosphate bond in 3 phosphoglycerate transformed into a high energy phosphate bond that can be transferred to ADP?
9. what is the final reaction of glycolysis?
10. what is the total free energy change from glycolysis?
11. why does NADH need to be reoxidized and what are the two pathways in which this is accomplished?
12. what are the two fates for pyruvate?
13. why are reducing equivalent shuttles required?
14. describe the G3P shuttle.
15. describe the malate-aspartate shuttle.
16. describe anaerobic glycolysis.
17. what is the ATP production of the G3P vs. the malate-aspartate shuttle?
18. describe the difference of ATP production possible in aerobic vs. anaerobic glycolysis.
19. how is anaerobic glycolysis related to body pH?
20. when do cells or tissues use anaerobic glycolysis vs. aerobic?
answers
1. glycolysis is the splitting of glucose into pyruvate and production of 4 ATP molecules per molecule of glucose.
2. the preparative phase, in which glucose is phosphorylated twice by ATP and cleaved into two triose phosphates, and the ATP-generating phase, in which the triose phosphates produce 2 ATP each.
3. phosphorylation of glucose yields glucose-6-phosphate via hexokinases.
4. isomerization of glucose 6-phosphate via phosphoglucose isomerase to fructose 6-phosphate, phosphorylation to fructose 1,6 bisphosphate via phosphofructokinase-1 (PFK-1), and cleavage into two triose molecules (glyceraldehyde 3-phosphate) via aldolase.
5. G3P is oxidized by glyceraldehyde-3-phosphate dehydrogenase, which has a cysteine residue near the active site which accepts a high energy acyl phosphate and transfers it to G3P. in this process, NADH and a high energy phosphate bond are formed.
6. the formation of a high energy phosphate bond without oxygen via direct transfer of a phosphate bond from a high energy intermediate.
7. 1,3 bisphosphoglycerate transfers its phosphate group to ADP via phosphoglycerate kinase, forming 3 phosphoglycerate.
8. the low energy phosphate bond in 3-phosphoglycerate is first shifted to the middle carbon via phosphoglyceromutase, and then transformed into a higher energy phosphate bond via enolase, which removes a water molecule and turns the compound into phosphoenolasepyruvate.
9. transfer of the high energy phosphate bond phospho enol pyruvate to ADP, turning the compound into pyruvate.
10. -22kcal
11. NADH needs to be reoxidized in order to continually accept electrons during the oxidation of G3P via G3P dehydrogenase. when oxygen is available, reducing equivalents are shuttled across the mitochondrial membrane to the electron chain. when oxygen is not available, NADH transfers its electrons to pyruvate, which is then reduced to lactate.
12. pyruvate can be reduced to lactate in anaerobic glycolysis, or can be converted into acetyl coenzyme A and further oxidized in aerobic glycolysis.
13. because the mitochondrial membrane is impermeable to NADH.
14. the G3P shuttle is the main shuttle mechanism in which cytosolic NAD+ is regenerated by G3P dehydrogenase, which transfers the electrons from NADH2 to DHAP, forming G3P. G3P then diffuses across the mitochondrial membrane and donates its electrons to FAD, forming FADH2 and reforming DHAP, which can then diffuse back and be reused in the reaction.
15. the malate-aspartate shuttle is the other way that NAD+ is regenerated: NADH transfers its electrons to oxaloacetate, reducing it to malate, which is then transported across the mitochondrial membrane via a translocase. in the membrane, the malate reduces NAD to NADH2, which can then be used in the electron transport chain. the oxaloacetate that is formed is transaminated to aspartate, which is transported back across the mitochondrial membrane via another translocase, and transaminated back into oxaloacetate in the cytosol.
16. anaerobic glycolysis occurs when the the oxidative capacity of a cell is limited. in this reaction, the electrons from NADH2 are donated to pyruvate, creating lactic acid, catalyzed by the enzyme lactate dehydrogenase.
17. the G3P shuttle produces 1.5 moles of ATP per NADH2, whereas the malate-asparate shuttle produces 2.5 moles of ATP per NADH2.
18. in anaerobic glycolysis, pyruvate is reduced to lactate and thus cannot produce any more ATP besides the two moles (per glucose) that is produced during glycolysis. in aerobic glycolysis, in addition to these two ATP's, the pyruvate is oxidized and used in the mitochondria to produce 25 ATP (per glucose), and the NADH produced can add another 3 (if using the G3P pathway) or 5 (if using the malate aspartate pathway) ATP molecules.
19. lactic acid is produced from the reduction of pyruvate in anaerobic glycolysis via lactate dehydrogenase. at the normal intracellular pH of 7.35, lactic acid dissociates into lactate and H+, which is then transferred out of the cell and eventually into the blood, where it can influence body pH.
20. tissues that have a low ATP demand and low O2 availability. also, blood cells have undergo anaerobic glycolysis because aerobic metabolism might interfere with the loading and unloading of O2 from hemoglobin. also, in other tissues with mitochondria and O2 availability, anaerobic glycolysis is used when the cell's energetic demands are not met by aerobic processes.
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