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Chapter 18 Glycolysis
Dr Khairul Ansari
Chapter 18
Living organisms, like machines, conform to the law of conservation of energy, and must pay for all their activities in the currency of catabolism. Ernest Baldwin Dynamic Aspects of Biochemistry
Louie Pasteur s scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis.
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Essential Question
What is the chemical basis and logic for glycolysis, the central pathway of metabolism; that is, how does glycolysis work? What are the essential features of glycolysis? Why are coupled reactions important in glycolysis? What are the chemical principles and features of the first phase of glycolysis? What are the chemical principles and features of the second phase of glycolysis? What are the metabolic fates of NADH and pyruvate produced in glycolysis? How do cells regulate glycolysis? Are substrates other than glucose used in glycolysis? How do cells respond to hypoxic stress?
Gycolysis
Living organism appears in O2 free environment Gycolysis does not need O2 Gycolysis plays significant roles in anaerobic metabolism in the first 2 billion years of evolution Otto Warburg, Gustav Embden and Otto Fritz Meyerhof- worked out of the glycolysis pathway Gycolysis is also known as Embden-Meyerhof (or Warburg) Pathway
Brain, Kidney medulla and rapidly contracting skeleting muscle and some cells such as erythrocytes and sperm use glucose as only energy source.
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Glycolysis
Essentially all cells carry out glycolysis Ten reactions essentially the same in all cells but with different rates Two phases: First phase converts glucose to two glyceraldehyde-3-P Second phase produces two pyruvates Products are pyruvate, ATP and NADH Three possible fates for pyruvate Under aerobic conditions pyruvate can be converted to acetyl-CoA that enter TCA cycle It can also be converted into glucose via gluconeogenesis. Under anaerobic conditions, it may be converted into lactic acid. In yeast, pyruvate is converted into ethanol instead.
Figure 18.2 Pyruvate produced in glycolysis can be used by cells in several ways. In animals, pyruvate is normally converted to acetylcoenzyme A, which is then oxidized in the TCA cycle to produce CO2. When oxygen is limited, pyruvate can be converted to lactate. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.
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What Are the Chemical Principles and Features of the First Phase of Glycolysis?
The first reaction - phosphorylation of glucose Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Be sure you can interconvert Keq and standard-state free energy change Be sure you can use Eq. 3.13 to generate the values shown in the far right column of Table 18.1. -D-Glucose + ATP4- -D-Glucose -6-phosphate2- +ADP3- + H+ G = -16.7 KJ/mol Phosphorylation of glucose cost 13.8 KJ/mol
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Rxn 1: Hexokinase
1st step in glycolysis; G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell Km for glucose is 0.1 mM; cell has 4 mM glucose So hexokinase is normally active! Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why? Hexokinase reaction is one of the three points in glycolysis pathway that are regulated Mg2+ is required (the true substrate is MgATP2-) There are two isozymes of Hexokinase (type I and type II)
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Rxn 1: Hexokinase
Km for glucose is 0.03 mM for type I hexokinase and 0.3 mM for type II hexokinase Type I is primary hexokinase in brain Type IV (glucokinase) is predominant in liver- doest not get inhibited by the product (Km is approximately 10 mM) When glucose level is high the liver hexokinase (glucokinase) phosphorylates glucose and eventually converts to glycogen Glucokinase is inducible (by insulin) and acts when level of glucose is very high. In diabetes mellitus (patient producing insufficient insulin) level of glucokinase is less.
These steady-state concentrations are used to obtain the cellular values of G found in Table 18.1 and Figure 18.22.
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Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.
Reaction 1: Hexokinase
Figure 18.6 The (a) open and (b) closed states of yeast hexokinase. Binding of glucose (green) induces a conformation change that closes the active site, as predicted by Daniel Koshland. The induced fit model for enzymes is discussed on page 409 of the text.
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Reaction 2: Phosphoglucoisomerase
Glucose-6-P to Fructose-6-P Why does this reaction occur? next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH isomerization activates C-3 for cleavage in aldolase reaction Ene-diol intermediate in this reaction
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Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)
Reaction 3: Phosphofructokinase
PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, negative G - means PFK is highly regulated ATP inhibits, AMP reverses inhibition Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high The reaction is endegonic by itself (G = 16.6 KJ/mol), but exergonic when coupled with ATP hydrolysis (G = -14.2 KJ/mol
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Phosphofructokinase is the second priming reaction of glycolysis. ATP is consumed in this priming reaction, so that more ATP can be produced further along the pathway.
Phosphofructokinase
Phosphofructokinase is the valve controlling rate of glycolysis ATP is both substrate and allosteric regulator of PFK High ATP turn off glycolysis in cytosol. ATP level does not fluctuate to greater extend in most tissue ADP + ADP ATP + AMP Small drop of ATP level in cell results a large increase in AMP AMP reverse the PFK inhibition by ATP PFK activity is high when energy status of cell is low, and PFK activity is low when energy status is high. Fructose-2,6-bisphosphate also regulatesPFK Citrate an intermediate of TCA cycle is also an allosteric inhibitor of PFK (PFK activity coupled with TCA cycle)
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Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid.
Phosphofructokinase is regulated by fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-P.
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Figure 18.11 F-2,6-BP stimulates PFK by decreasing the inhibitory effects of ATP.
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Triose phosphate isomerase completes the first phase of glycolysis. Each glucose has been converted to two molecules of glyceraldehyde3-phosphate.
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What Are the Chemical Principles and Features of the Second Phase of Glycolysis?
Metabolic energy of glucose produces 4 ATP Net ATP yield for glycolysis is two ATP The second phase of glycolysis involves two very high-energy phosphate intermediates 1,3-bisphosphoglycerate (1,3-BPG) Phosphoenolpyruvate (PEP)
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G3P-DH
G3P-OH can be inactivated by reaction with iodoacetate, which reacts with and block the essential cysteine sulfhydryl
Arsenate is a substrate for the G3P-DH reaction, forming 1-arseno-3-phosphoglycerate. This product breaks down to 3-phosphoglycerate, essentially bypassing the phosphoglycerate kinase reaction. The result is that glycolysis in the presence of arsenate produces no net ATP.
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Phosphoglycerate kinase transfers a phosphoryl group from 1,3bisphosphoglycerate to ADP to form ATP. This type of ATPsynthesizing reaction is referred to as a substrate-level phosphorylation
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The open (a) and closed (b) forms of phosphoglycerate kinase. ATP (cyan), 3phosphoglycerate (purple), and Mg2+ (gold).
2,3-BPG is made by reactions that detour around the phosphoglycerate kinase reaction
2,3-bisphosphoglycerate is an important regulator of hemoglobin (see pages 505-506 of the text) 2,3-BPG is formed from 1,3-BPG by bisphosphoglycerate mutase 3-phosphoglycerate is then formed by 2,3bisphosphoglycerate phosphatase Most cells contain only a trace of 2,3-BPG, but erythrocytes typically contain 4-5 mM 2,3-BPG
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2,3-BPG is made by reactions that detour around the phosphoglycerate kinase reaction
2,3-BPG is made by reactions that detour around the phosphoglycerate kinase rxn
Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3phosphoglycerate.
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Phosphoglycerate mutase catalyzes a phosphoryl group transfer from C-3 to C-2 Rationale for this reaction in glycolysis: It repositions the phosphate to make PEP in the following reaction (enolase) Note the phospho-histidine intermediates Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2,3-BPG is required to phosphorylate His Nomenclature note: a mutase catalyzes migration of a functional group within a substrate
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Reaction 9: Enolase
Conversion of 2-Phosphoglycerate to PEP The enolase makes a high-energy phosphate in preparation for ATP synthesis in step 10 The overall G for this reaction is 1.8 kJ/mol How can such a reaction create a PEP? "Energy content" of 2-PG and PEP are similar Enolase just rearranges 2-PG to a form that releases more energy upon hydrolysis
Reaction 9: Enolase
The enolase reaction creates a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis.
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The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP. Note that two ATP are produced here, since two PEP are formed from one glucose. These two ATP are the payoff of glycolysis.
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Figure 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.
18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?
NADH can be recycled via aerobic or anaerobic pathways
NADH represents energy - two possible fates: If O2 is available (aerobic conditions), NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation In anaerobic conditions, NADH is oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis
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18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?
Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (Right) Fermentation at a bourbon distillery. A mash of corn and other grains is fermented by yeast, producing ethanol and CO2, which can be seen bubbling to the surface.
18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?
Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction. Hibernating turtles, trapped beneath ice and lying in mud, become anoxic and convert glucose mainly to lactate. Their shells release minerals to buffer the lactate throughout the period of hibernation.
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18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?
In aerobic conditions, pyruvate proceeds through the tricarboxylic acid (TCA) cycle (see Chapter 19) Anaerobic metabolism of pyruvate leads to lactate (in microorganisms and animals) or ethanol (in yeast) These are examples of fermentation the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons
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Otto Warburg
Otto Warburg, Nobel Prize in Physiology or Medicine, 1931 for his discovery of the nature and mode of action of the respiratory enzyme (cytochrome oxidase see Ch. 20)
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The elegant evidence of regulation See Figure 18.22 Standard state G values are scattered, with both plus and minus values and no apparent pattern The plot of G values in cells is revealing: Most values near zero 3 of 10 reactions have large, negative G These 3 reactions with large negative G are sites of regulation (HK, PFK, PK) Regulation of these three reactions can turn glycolysis off and on
Figure 18.22 The free energies of the reactions of glycolysis under standardstate conditions.
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Figure 18.22 The free energies of the reactions of glycolysis under actual intracellular concentrations of metabolites in erythrocytes.
Tumors show very high rates of glycolysis, as shown by Otto Warburg early in the 20th century This observation is the basis of tumor detection by positron emission tomography (PET) Metabolites labeled with 18F can be taken up by human cells (in the brain, for example) Decay of 18F results in positron emission Positron-electron collisions produce gamma rays Detection with gamma ray cameras provides 3D models of tumor extent and location 2-[18F]fluoro-2-deoxy-glucose, used for this purpose, is a substrate for hexokinase
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Decay of 18F results in positron emission. Positron-electron collisions produce gamma rays
Detection with gamma ray cameras provides 3D models of tumor extent and location
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Sugars other than glucose can be glycolytic substrates Fructose, mannose and galactose can all be used Fructose and mannose are routed into glycolysis by fairly conventional means. See Figure 18.23 Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... See Figure 18.24
Figure 18.23 Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway.
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Figure 18.25 The galactose-1-phosphate uridylyltransferase reaction involves a ping-pong kinetic mechanism.
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UDP-glucose pyrophosphorylase uses Gal-1-P, reducing galactose toxicity in adults Figure 18.26
Glycerol is produced in the decomposition of triacylglycerols. It can be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase.
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Glycerol is produced in the decomposition of triacylglycerols. It can be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then oxidized to dehydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase.
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VHL is the von Hippel Lindau subunit of the ubiquitin E3 ligase that targets proteins for proteasome degradation
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Figure 18.28
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Chapter 19
Thus times do shift, each thing his turn does hold; New things succeed, as former things grow old. Robert Herrick Hesperides (1648)
A time-lapse photograph of a ferris wheel at night. Aerobic cells use a metabolic wheel the TCA cycle to generate energy by acetyl-CoA oxidation.
How is pyruvate oxidized under aerobic conditions, and what is the chemical logic that dictates how this process occurs?
Essential Questions
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Outline
What is the chemical logic of the TCA cycle? How is pyruvate oxidatively decarboxylated to acetyl-CoA? How are two CO2 molecules produced from acetyl-CoA? How is oxaloacetate regenerated to complete the TCA cycle? What are the energetic consequences of the TCA cycle? Can the TCA cycle provide intermediates for biosynthesis? What are the anaplerotic, or filling up, reactions? How is the TCA cycle regulated? Can any organisms use acetate as their sole carbon source?
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4 electrons are removed as NADH in glycolysis 2 NADH are produced during decarboxylation of two molecules of pyruvate For each acetyl-CoA oxidized in TCA cycle, 8 more electrons are removed as NADH (3) and FADH2(1) H3CCOO- + 2H20 + H+ 2CO2 + 8H In electron transport pathway these electrons combines with O2 to produce water 8H + 2O2 H2O Net reaction of TCA cycle and electron transport pathway is H3CCOO- + 2O2 + H+ 2CO2 + 2H2O
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Acetyl-CoA- entry point of new carbon into the TCA cycle Generally (in biological system) C-C occur cleavage between a and carbon Or between two carbons As acetate does not have a carbon neither of these cleavage are possible. Condensation of acetate with oxaloacetate facilitate cleavage
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The TCA cycle seems like a complicated way to oxidize acetate units to CO2 What are the possibilities (based on our knowledge of metabolism)? cleavage between Cs and to a carbonyl -cleavage of an -hydroxyketone These ways to cleave C-C bonds don't work for acetylCoA
This type of cleavage occurs in the fructose bisphosphate aldolase reaction in glycolysis But it cant be used for acetate, which does not have a carbon
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-Cleavage of an -Hydroxyketone
This type of cleavage occurs in the transaldolase reaction (described in Chapter 22). But it would require hydroxylation of acetate, which is not a favorable or facile reaction for acetate Living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a -cleavage TCA combines this cleavage with oxidation to form CO2, regenerating oxaloacetate and capturing all the energy as NADH and ATP!
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Decarboxylation of pyruvate yields hydroxyethyl-TPP. Transfer to lipoic acid is followed by formation of acetyl-CoA.
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Figure 19.6 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. NADH is an allosteric inhibitor of citrate synthase
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Figure 19.7 Citrate synthase in mammals is a dimer of 49-kD subunits. In the monomer shown here, citrate (blue) and CoA (red) bind to the active site, which lies in a cleft between two domains and is surrounded mainly by -helical segments.
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Figure 19.8 The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate.
Figure 19.8 The active site of aconitase. The iron-sulfur cluster (pink) is coordinated by cysteines (orange) and isocitrate (purple).
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Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle in vivo, although it has no apparent effect on any of the isolated TCA cycle enzymes. The action of aconitase has been traced to aconitase Aconitase is inhibited by fluorocitrate, which is formed from fluoracetate in two steps, as shown here.
Oxidative decarboxylation of isocitrate yields -ketoglutarate This is classic NAD+ chemistry (hydride removal) followed by a decarboxylation Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH The mechanism is typical of NAD+-dependent enzymes (see Figure 19.10) The reaction involves (first) oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate, then a -decarboxylation reaction that expels the central carboxyl group as CO2
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Figure 19.10 The isocitrate dehydrogenase reaction. Hydride removal by NAD+ is followed by a -decarboxylation reaction that expels the central carboxyl group as CO2
-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle
A second oxidative decarboxylation This enzyme is nearly identical to pyruvate dehydrogenase structurally and mechanistically Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD You know the mechanism if you remember pyruvate dehydrogenase
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-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle
Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a multienzyme complex consisting of -ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. The complex uses five different coenzymes.
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Hydrolysis of succinyl-CoA (a CoA ester) drives the phosphorylation of GDP to produce GTP
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Figure 19.13
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The dehydrogenases that require nicotinamide coenzymes are stereospecific, and they transfer hydride to either the pro-R or the pro-S positions selectively What accounts for this specificity? The enzymes involved are asymmetric structures. The nicotinamide coenzyme (and substrate) fit into the active site in only one way The hydride transfers can only be accomplished to or from one side or the other of the NAD+ molecule Dehydrogenases (and other enzymes too) are also stereospecific with respect to the substrates as well
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The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle
The carbonyl C of acetyl-CoA becomes CO2 only in the second turn of the cycle (following entry of acetyl-CoA ) The methyl C of acetyl-CoA survives two cycles completely, but half of what's left exits the cycle on each turn after that The C-C bond cleaved in a given TCA cycle actually entered as an acetate in the previous turn Thus the oxidative decarboxylations that cleave this bond are just a disguised acetate C-C cleavage and oxidation
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The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle
Figure 19.15 The fate of the carbon atoms of acetate in successive TCA cycles.
The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle
Figure 19.15 The fate of the carbon atoms of acetate in successive TCA cycles.
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Figure 19.17 Pyruvate carboxylase (shown here), and also phosphoenolpyruvate (PEP) carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.
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Figure 19.17 Pyruvate carboxylase , phosphoenolpyruvate (PEP) carboxylase (shown here), and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.
Figure 19.17 Pyruvate carboxylase, and also phosphoenolpyruvate (PEP) carboxylase and malic enzyme (shown here) catalyze anaplerotic reactions, replenishing TCA cycle intermediates.
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This reaction *might* be an anaplerotic reaction, except for the fact that CO2 binds weakly to PEP carboxykinase, and oxaloacetate binds very tightly. As a result, the enzyme favors formation of PEP from oxaloacetate. Thus the reaction operates in the wrong direction to be an anaplerotic reaction.
The pancreas releases insulin in response to an increase of blood glucose What cellular processes mediate this response? It was long accepted that ATP produced by catabolism activated K+ channels in the plasma membrane of -cells in the pancreas However, recent research has shown that anaplerotic enzymes feed alternative pathways that produce cytosolic signal molecules that also support insulin secretion
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The pyruvate/malate cycle in pancreatic -cells. Oxaloacetate produced by high levels of pyruvate carboxylase is converted to malate, then exported to the cytosol.
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Figure 19.19 Regulation of the pyruvate dehydrogenase reaction. Phosphorylation inactivates; Dephosphorylation activates.
19.10 Can Any Organisms Use Acetate as Their Sole Carbon Source?
The Glyoxylate Cycle Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA The glyoxylate cycle offers a solution for plants and some bacteria and algae The CO2-evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the short-circuiting enzymes
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19.10 Can Any Organisms Use Acetate as Their Sole Carbon Source?
Figure 19.20 The glyoxylate cycle. The first two steps are identical to TCA reactions. Isocitrate lyase and malate synthase short-circuit the TCA cycle, forming malate and succinate from isocitrate and another acetyl-CoA.
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Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate
Figure 19.21 The isocitrate lyase reaction. Isocitrate lyase catalyzes an aldol cleavage and is similar to the aldolase reaction in glycolysis.
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Figure 19.20 Glyoxysomes lack three of the enzymes needed to run the glyoxylate cycle. Succinate dehydrogenase, fumarase, and malate dehydrogenase are all borrowed from mitochondria.
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