BCH Graduate Survey of Biochemistry

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1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham Lecture 50 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 16 The Citric Acid Cycle 2008 W. H. Freeman and Company

3 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.

4 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?

5 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 CO 2 is removed by a second round of oxidative decarboxylation by the action of α-ketoglutarate dehydrogenase to yield succinyl-coa and NADH.

6 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 FADH 2 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.

7 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.

10 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?

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

12 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?

15 Anaerobic bacteria contain an incomplete citric acid cycle.

16 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?

18 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.

19 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.

This enzyme is controlled by phosphorylation/dephos phorylation.

20 The citric acid cycle and the glyoxylate cycles are coordinately regulated. The major regulatory step is isocitrate dehydrogenase of the citric acid cycle. This enzyme is controlled by phosphorylation/dephos phorylation. 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.

21 Theoretical efficiency of complete oxidation of glucose to form ATP equivalents in aerobic conditions Glucose + 6O 2 6CO 2 + 6H 2 O ATP + H 2 O ADP + P i + H + G = 2872 kj/mol ( kcal/mol) G = 30.5 kj/mol ( 7.3 kcal/mol) Glycolysis (net) Glucose + 2NAD + + 2ADP + 2P i 2Pyruvate + 2NADH + 2ATP + 2H 2 O 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 kcal/mol = 2.12%.

22 Pyruvate Dehydrogenase (net) 2Pyruvate + 2CoA + 2NAD + 2Acetyl-CoA + 2NADH + 2CO 2 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 kcal = 0.0%). Citric Acid Cycle (net) 2Acetyl-CoA + 2ADP + 6NAD + + 2FAD + 4CO 2 + 2ATP + 6NADH + 2FADH Energy content produced in ATP equivalents (22 where NADH = 3ATPs and FADH = 2ATPs) is 671 kj/mol or kcal/mol 23.3% (overall efficiency for actual ATP produced 61 kj or 14.6 kcal/ 2872 kj or kcal = 2.1%). Total of 4ATPs made from glucose so far is 4.25% of energy in glucose.

23 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 = 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 X 30.5 kj = 976 kj/mol or 32 X 7.3 kcal = 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.

24 Compare the theoretical efficiency of % 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. 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? Source:

25 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 O 2 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.

26 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?

TCA CYCLE (Citric Acid Cycle)

TCA CYCLE (Citric Acid Cycle) TCA CYCLE The Citric Acid Cycle is also known as: Kreb s cycle Sir Hans Krebs Nobel prize, 1953 TCA (tricarboxylic acid) cycle The citric acid cycle requires aerobic conditions!!!!