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BCH 5045

Graduate Survey of Biochemistry

Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham

Lecture 50

Slide sets available at: http://hort.ifas.ufl.edu/teach/guyweb/bch5045/index.html

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• LEHNINGER • PRINCIPLES OF BIOCHEMISTRY

• Fifth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 16 The Citric Acid Cycle



Evidence accumulated in recent years suggests that in plants, there are conditions where the flux as shown to the right is not entirely cyclic, but that non-cyclic metabolic activities of parts of the Citric Acid Cycle seem to have important biological functions.

Presenter

Presentation Notes

FIGURE 16-13 Products of one turn of the citric acid cycle. At each turn of the cycle, three NADH, one FADH2, one GTP (or ATP), and two CO2 are released in oxidative decarboxylation reactions. Here and in several following figures, all cycle reactions are shown as proceeding in one direction only, but keep in mind that most of the reactions are reversible (see Figure 16-7).



Note the structure of citrate, three carboxyl groups on a three carbon backbone and on the middle carbon, there is an OH group. Is citrate a chiral compound?

Presenter

Presentation Notes

FIGURE 16-7 Reactions of the citric acid cycle. The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as CO2 in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The number beside each reaction step corresponds to a numbered heading on pages 622–628. The red arrows show where energy is conserved by electron transfer to FAD or NAD+, forming FADH2 or NADH + H+. Steps 1, 3, and 4 are essentially irreversible in the cell; all other steps are reversible. The product of step 5 may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst.



Acetyl-CoA generated by the pyruvate dehydrogenase complex condenses with oxaloacetate to form the first product of the cycle, citric acid, catalyzed by the action of citrate synthase. Citric acid then undergoes a dehydration to form cis-aconitate which is then rehydrated to form isocitric acid. This reaction is catalyzed by the enzyme aconitase. Isocitrate is oxidatively decarboxylated reducing NAD+ to NADH and forming α-ketoglutarate. A second CO2 is removed by a second round of oxidative decarboxylation by the action of α-ketoglutarate dehydrogenase to yield succinyl-CoA and NADH.



The energy available in the thioester linkage of succinyl-CoA is used to produce GTP/ATP, CoASH and succinate. Succinate undergoes dehydrogenation (oxidation) catalyzed by the action of succinate dehydrogenase to form FADH2 and fumarate. Water is added to fumarate to form malate. Malate is oxidized by the action of malate dehydrogenase forming NADH and oxaloacetate regenerating the original acceptor for acetyl-CoA and ready to begin another round of the cycle.



Adding and removing water is a common strategy for rearranging metabolites that happen to have adjacent carbon atoms with a hydroxyl group and a hydrogen.



Presenter

Presentation Notes

MECHANISM FIGURE 16-11 Isocitrate dehydrogenase. In this reaction, the substrate, isocitrate, loses one carbon by oxidative decarboxylation. See Figure 14-13 for more information on hydride transfer reactions involving NAD+ and NADP+.



Presenter

Presentation Notes

FIGURE 16-12a The succinyl-CoA synthetase reaction. (a) In step 1 a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. In step 2 the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. In step 3 the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP). FIGURE 16-12b The succinyl-CoA synthetase reaction. (b) Active site of succinyl-CoA synthetase of E. coli (derived from PDB ID 1SCU). The active site includes part of both the α (blue) and the β (brown) subunits. The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of P–His246 in the α chain, stabilizing the phosphohistidyl enzyme. The bacterial and mammalian enzymes have similar amino acid sequences and three-dimensional structures.



So why can’t malonate form a carbon-carbon double bond? What kind of inhibitor would you think malonate is of the succinate DH reaction?



Will maleate, a cis-trans isomer of fumarate, be converted to malate by the fumarase?



Given the ΔG´° the reaction is positive as written, what conditions would be needed to drive the oxidation of malate to form OAA and NADH? Under what circumstances would you expect the conditions needed to drive the direction of the reaction towards OAA?



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Presentation Notes

FIGURE 16-18 Regulation of metabolite flow from the PDH complex through the citric acid cycle in mammals. The PDH complex is allosterically inhibited when [ATP]/[ADP], [NADH]/[NAD+], and [acetyl-CoA]/[CoA] ratios are high, indicating an energy-sufficient metabolic state. When these ratios decrease, allosteric activation of pyruvate oxidation results. The rate of flow through the citric acid cycle can be limited by the availability of the citrate synthase substrates, oxaloacetate and acetyl-CoA, or of NAD+, which is depleted by its conversion to NADH, slowing the three NAD-dependent oxidation steps. Feedback inhibition by succinyl-CoA, citrate, and ATP also slows the cycle by inhibiting early steps. In muscle tissue, Ca2+ signals contraction and, as shown here, stimulates energy-yielding metabolism to replace the ATP consumed by contraction.



Presenter

Presentation Notes

TABLE 16-1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation



Anaerobic bacteria contain an incomplete citric acid cycle.

Presenter

Presentation Notes

FIGURE 16-14 Biosynthetic precursors produced by an incomplete citric acid cycle in anaerobic bacteria. These anaerobes lack α-ketoglutarate dehydrogenase and therefore cannot carry out the complete citric acid cycle. α-Ketoglutarate and succinyl-CoA serve as precursors in a variety of biosynthetic pathways. (See Figure 16-13 for the "normal" direction of these reactions in the citric acid cycle.)



The Citric acid cycle functions as an anabolic process as well as a catabolic process. But what happens if some of the intermediates are used up and not available to regenerate OAA?

Presenter

Presentation Notes

FIGURE 16-15 Role of the citric acid cycle in anabolism. Intermediates of the citric acid cycle are drawn off as precursors in many biosynthetic pathways. Shown in red are four anaplerotic reactions that replenish depleted cycle intermediates (see Table 16-2)



Presenter

Presentation Notes

FIGURE 16-20 Glyoxylate cycle. The citrate synthase, aconitase, and malate dehydrogenase of the glyoxylate cycle are isozymes of the citric acid cycle enzymes; isocitrate lyase and malate synthase are unique to the glyoxylate cycle. Notice that two acetyl groups (pink) enter the cycle and four carbons leave as succinate (blue). The glyoxylate cycle was elucidated by Hans Kornberg and Neil Madsen in the laboratory of Hans Krebs. FIGURE 16-21 Electron micrograph of a germinating cucumber seed, showing a glyoxysome, mitochondria, and surrounding lipid bodies.



The glyoxylate cycle involves several compartments in higher cells; a lipid body (in the cytosol), a specialized organelle known as the glyoxysome, and the mitochondrion. Fatty acids are transferred from a lipid body to the glyoxysome where they are broken down by β-oxidation in to acetyl-CoA units. OAA in the mitochondrion is converted to aspartate which is translocated to the glyoxysome where it is deaminated to regenerate OAA. The OAA condenses with acetyl-CoA to form citrate by the action of citrate synthase (but not the citrate synthase in the mitochondrion. The citrate is transformed into isocitrate by aconitase.



The isocitrate is next split by isocitrate lyase into glyoxylate and succinate. The succinate is returned to the mitochondrion to be used in the citric acid cycle to produce OAA. The glyoxylate formed by the splitting of isocitrate in the glyoxysome condenses with acetyl-CoA by the action of malate synthase to form malate. Malate is oxidized by malate dehydrogenase to form OAA. The OAA so formed is a substrate for PEP-carboxykinase which in the presence of GTP yields PEP. The PEP is then used in gluconeogenesis.



The major regulatory step is isocitrate dehydrogenase of the citric acid cycle. This enzyme is controlled by phosphorylation/dephosphorylation. In the phosphorylated state IDH is inhibited and isocitrate flows into the glyoxylate cycle. When dephosphorylated, IDH is active and isocitrate is metabolized by the citric acid cycle. Further, the allosteric activators of IDH are inhibitors of isocitrate lyase.

The citric acid cycle and the glyoxylate cycles are coordinately regulated.

Presenter

Presentation Notes

FIGURE 16-22 Relationship between the glyoxylate and citric acid cycles. The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously with, and mesh with, those of the citric acid cycle (in mitochondria), as intermediates pass between these compartments. The conversion of succinate to oxaloacetate is catalyzed by citric acid cycle enzymes. The oxidation of fatty acids to acetyl-CoA is described in Chapter 17; the synthesis of hexoses from oxaloacetate is described in Chapter 20. FIGURE 16-23 Coordinated regulation of glyoxylate and citric acid cycles. Regulation of isocitrate dehydrogenase activity determines the partitioning of isocitrate between the glyoxylate and citric acid cycles. When the enzyme is inactivated by phosphorylation (by a specific protein kinase), isocitrate is directed into biosynthetic reactions via the glyoxylate cycle. When the enzyme is activated by dephosphorylation (by a specific phosphatase), isocitrate enters the citric acid cycle and ATP is produced.



Theoretical efficiency of complete oxidation of glucose to form ATP equivalents in aerobic conditions

Glucose + 6O2 → 6CO2 + 6H2O ∆G°′ = –2872 kj/mol (–686.5 kcal/mol)

ATP + H2O → ADP + Pi + H+ ∆G°′ = –30.5 kj/mol (–7.3 kcal/mol)

Glycolysis (net)

Glucose + 2NAD+ + 2ADP + 2Pi → 2Pyruvate + 2NADH + 2ATP + 2H2O

∆G°′ = –85 kj/mol (–20.3 kcal/mol)

Energy content produced in theoretical ATP equivalents (6, where NADH = 3ATPs) is –183 kj/mol or –43.8 kcal/mol ∼6.38%. However, overall efficiency for actual ATP produced –61 kj or –14.6 kcal ÷ –2872 kj or –686.5 kcal/mol = ∼2.12%.



Pyruvate Dehydrogenase (net)

2Pyruvate + 2CoA + 2NAD+ → 2Acetyl-CoA + 2NADH + 2CO2

∆G°′ = –33.4 kj/mol (–8.0 kcal/mol) ∴Total net per mol of glucose is –66.8 kj or –16 kcal.

Energy content produced in theoretical ATP equivalents (6) is –183 kj/mol or –43.8 kcal/mol ∼6.4% (overall efficiency for actual ATP produced –0 kj or –0 kcal ÷ –2872 kj or –686.5 kcal = ∼0.0%).

Citric Acid Cycle (net)

2Acetyl-CoA + 2ADP + 6NAD+ + 2FAD+ → 4CO2 + 2ATP + 6NADH + 2FADH

Energy content produced in ATP equivalents (22 where NADH = 3ATPs and FADH = 2ATPs) is –671 kj/mol or –160.6 kcal/mol ∼23.3% (overall efficiency for actual ATP produced –61 kj or –14.6 kcal/–2872 kj or –686.5 kcal = ∼2.1%).

Total of 4ATPs made from glucose so far is 4.25% of energy in glucose.



Total maximum theoretical yield of ATP is 38 for Glycolysis-PDH-Citric Acid Cycle, but real output is only 4ATPs or 4.25%.

38 X –30.5 kj = –1159 kj/mol or 38 X –7.3 kcal = –277.4 kcal/mol (overall efficiency 40.4%).

Alternatively the text uses 2.5 ATP equivalents per NADH, and 1.5 ATP equivalents per FADH oxidized. So the theoretical yield of ATP is 32 and not 38.

32 X –30.5 kj = –976 kj/mol or 32 X –7.3 kcal = –233.6 kcal/mol (overall efficiency 34.0%).

So what makes it possible to go from 4ATPs produced to 32ATPs?

So what biochemical and cellular factors make the 40.4 or 34.0% efficiency only a theoretical estimation and not the real efficiency?

Where is the other ~60-65% of the energy content of glucose going?



Compare the theoretical efficiency of 34 - 40.4% for the complete oxidation of glucose in biological systems in the presence of oxygen versus 14%–26% for the energy from the fuel you put in your tank that is used to move your car down the road or run useful accessories, such as air conditioning. The rest of the energy is lost to engine and driveline inefficiencies and idling.

Source: http://www.fueleconomy.gov/feg/atv.shtml

Would you not think that the potential to improve or double fuel efficiency with advanced technologies is enormous? Why are hybrids and electric cars so much more efficient than standard cars?

http://www.fueleconomy.gov/feg/atv.shtml�



The internal combustion engine involves an exothermic reaction that creates gases at high temperature and pressure, which are permitted to expand as a result of the combustion of fuel and an oxidizer O2 that occurs in a confined space. Work is performed by the expanding hot gases acting directly to cause movement in the engine, by acting on pistons and crank shaft to drive movement through the transmission to the wheels.



For gasoline-powered vehicles, more than 62% of the energy of combustion available is lost. Internal-combustion engines are very inefficient at converting the fuel's chemical energy to mechanical energy, losing energy to engine friction, pumping air into and out of the engine, and as wasted heat.

In urban driving, energy is lost to idling at stoplights or in traffic. Air conditioning, power steering, windshield wipers, and other accessories use energy generated from the engine. Improvements of fuel economy up to 1% may be achievable with more efficient alternator systems and power steering pumps. Energy is lost in the transmission and other parts of the driveline.

In us, where is the excess free energy in glucose going?

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