Chapter 10

1. Chapter 10 The Citric Acid Cycle The Citric Acid Cycle The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO2. A pathway providing many precursors for biosynthesis.

2. 1.The cellular respiration (complete oxidation of fuels) can be divided into three stages Stage I All the fuel molecules are oxidized to generate a common two-carbon unit, acetyl-CoA. Stage II The acetyl-CoA is completely oxidized into CO2, with electrons collected by NAD and FAD via a cyclic pathway (named as the citric acid cycle, Krebs cycle, or tricarboxylic acid cycle). Stage III Electrons of NADH and FADH2 are transferred to O2 via a series carriers, producing H2O and a H+ gradient, which will promote ATP formation. The Citric Acid Cycle

3. 1.The cellular respiration (complete oxidation of fuels) can be divided into three stages The Citric Acid Cycle Mitochondria is the major site for fuel oxidation to generate ATP.

4. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex Pyruvate is first transported into mitochondria via a specific transporter on the inner membrane. Pyruvate is converted to acetyl-CoA and CO2 by oxidative decarboxylation. The pyruvate dehydrogenasecomplex is a huge multimeric assembly of three kinds of enzymes, having 60 subunits in bacteria and more in mammals. Pyruvate is first decarboxylated after binding to the prosthetic group (辅基，TPP) of pyruvate dehydrogenase (E1), forming hydroxyethyl-TPP.（羟乙基-焦磷酸硫胺素） The Citric Acid Cycle

5. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The hydroxyethyl group attached to TPP is oxidized and transferred: First two electrons, then the acetyl group formed are all transferred to the lipoyllysyl (硫辛酰赖氨酰）group of dihydrolipoyl transacetylase (二硫辛酰转乙酰基酶，E2). The lipoyllysyl group serves as both electron and acetyl carriers. The acetyl group is then transferred (still catalyzed by E2) from acetyllipoamide乙酰硫辛酰胺to CoA-SH, forming acetyl-CoA. The oxidized lipoamide group is then regenerated by the action of dihydrolipoyl dehydrogenase (二硫辛酰脱氢酶，E3), with electrons collected by FAD and then by NAD+. The Citric Acid Cycle

6. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex Substrates of the five reactions catalyzed by pyruvate dehydrogenase complex are efficiently channeled : The lipoamide group attached to E2 swings between E1 (accepting the electrons and acetyl group) and E3 (giving away the electrons), passing the acetyl group to Coenzyme A on E2 The multienzyme complexes catalyzing the oxidative decarboxylation of a few different kinds of α-keto acids, pyruvate dehydrogenase complex, α -ketoglutarate（酮戊二酸） dehydrogenase complex and branched chain a-keto acid dehydrogenase complex show remarkable structure and function relatedness (all have identical E3, similar E1 and E2). The Citric Acid Cycle

7. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle The oxidative decarboxylation of pyruvate in mitochondria: producing acetyl-CoA and CO2.

8. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle

9. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle Electron micrograph of pyruvate dehydrogenase complexes from E. coli

10. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle

11. pyruvate acetyl-CoA CO2 hydroxyethyl-TPP E3 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex E2 (dihydrolipoyl transacetylase): consisting the core, 24 subunits; E1 (pyruvate dehydrogenase): bound to the E2 core, 24 subunits; E3 (dihydrolipoyl dehydrogenase): bound to the E2 core, 12 subunits. (a protein kinase and phosphoprotein phosphatase, not shown here, are also part of the complex) The Citric Acid Cycle A model of the E. coli pyruvate dehydrognase complexshowing the three kinds of enzymes and the flexible lipoamide arms covalently attached to E2.

12. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The E2 core (a total of 24 subunits) forms a hollow cube. The Citric Acid Cycle X-ray structure of the E2 transacetylase core: Only four out of eight trimers are shown here.

13. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle The oxidative decarboxylation of pyruvate is catalyzed by a multiezyme complex: pyruvate dehydrogenase complex.

14. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle

15. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle With the help of TPP, pyruvate is decarboxylated: identical reaction as catalyzed by pyruvate decarboxylase.

16. Dihydrolipoyl The lipoyllysyl group serves as the electron and acetyl carriers 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle

17. 2. Pyruvate is oxidized to acetyl-CoA by the catalysis of pyruvate dehydrogenase complex The Citric Acid Cycle

18. 3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway O2 consumption and pyruvate oxidation in minced muscle tissues were found to be stimulated by some four-carbon dicarboxylic acids (Fumarate, succinate, malate and oxaloacetate, five- carbon dicarboxylic acid (a-ketoglutarate ), or six-carbon tricarboxylic acids (citrate, isocitrate, cis-aconitate). A small amount of any of these organic acids stimulates many folds of pyruvate oxidation! Malonate（丙二酸） inhibits pyruvate oxidation regardless of which active organic acid is added! The Citric Acid Cycle

19. 3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway Hans Krebs proposed the “citric acid cycle” for the complete oxidation of pyruvate in animal tissues in 1937 (he wrongly hypothesized that pyruvate condenses with oxaloacetate in his original proposal). The citric acid cycle was confirmed to be universal in cells by in vitro studies with purified enzymes and in vivo studies with radio isotopes (“radio isotope tracer experiments”). Krebs was awarded the Nobel prize in medicine in 1953 for revealing the citric acid cycle (thus also called the Krebs cycle). The Citric Acid Cycle

20. 3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway The Citric Acid Cycle

21. 4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle • The 4-carbon oxaloacetate (草酰乙酸) acts as the “carrier” for the oxidation. • The two carbons released as 2 CO2 in the first cycle of oxidation are not from the acetyl-CoA just joined. • The 8 electrons released are collected by three NAD+ and one FAD. • One molecule of ATP (or GTP) is produced per cycle by substrate-level phosphorylation. The Citric Acid Cycle

22. 4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle The Citric Acid Cycle

23. 4. The acetyl group (carried by CoA) is completely oxidized to CO2via the citric acid cycle The Citric Acid Cycle

24. 5. The citric acid cycle consists of eight successive reactions Step 1 The methyl carbon of acety-CoA joins the carbonyl carbon of oxaloacetate via aldol condensation to form citrate (柠檬酸); citroyl-CoA is a transiently intermediate but hydrolyzed immediately in the active site of citrate synthase; hydrolysis of the thioester bond releases a large amount of free energy, driving the reaction forward; large conformational changes occur after oxaloacetate is bound and after citroyl-CoA is formed, preventing the undesirable hydrolysis of acetyl-CoA. The Citric Acid Cycle

25. 5. The citric acid cycle consists of eight successive reactions Step 2 Citrate is isomerized into isocitrate (get the six-carbon unit ready for oxidative decarboxylation) via a dehydration step followed by a hydration step; cis-aconitate (顺乌头酸) is an intermediate during this transformation, thus the catalytic enzyme is named as aconitase, which contains a 4Fe-4S iron-sulfur center directly participating substrate binding and catalysis. Step 3 Isocitrate is first oxidized and then decarboxylated to form α-ketoglutarate (a-酮戊二酸); oxalosuccinate is an intermediate; two electrons are collected by NAD+; the carbon released as CO2 is not from the acetyl group joined; catalyzed by isocitrate dehydrogenase. The Citric Acid Cycle

26. 5. The citric acid cycle consists of eight successive reactions Step 4α-ketoglutarate undergoes another round of oxidative decarboxylation; decarboxylated first, then oxidized to form succinyl-CoA (琥珀酰辅酶A); again the carbon released as CO2 is not from the acetyl group joined; catalyzed by a-ketoglutarate dehydrogenase complex; reactions and enzymes closely resemble pyruvate dehydrogenase complex (with similar E1 and E2, identical E3). Step 5 Succinyl-CoA is hydrolyzed to succinate (琥珀酸或戊二酸); the free energy released by hydrolyzing the thioesterbond is harvested by a GDP or an ADP to form a GTP or an ATP by substrate-level phosphorylation; the reversible reaction is catalyzed by succinyl-CoA synthetase (or succinic thiokinase ，琥珀酸硫激酶); The Citric Acid Cycle

27. 5. The citric acid cycle consists of eight successive reactions acyl phosphate and phophohistidyl enzyme are intermediates; the active site is located at the interface of two subunits; the negative charge of the phospho-His intermediate is stabilized by the electric dipoles of two a helices (one from each subunit). Step 6 Succinate is oxidized to fumarate (延胡索酸或反丁烯二酸); catalyzed by a flavoprotein succinate dehydrogenase (with a covalently bound FAD and three iron-sulfur centers), which is tightly bound to the inner membrane of mitochondria; malonate (丙二酸) is a strong competitive inhibitor of the enzyme, that will block the whole cycle. The Citric Acid Cycle

28. 5. The citric acid cycle consists of eight successive reactions Step 7 Fumarate （延胡索酸） is hydrated to L-malate（苹果酸）by the action of fumarase （延胡索酸酶）; the enzyme is highly stereospecific, only act on the trans and L isomers, not on the cis and D isomers (maleate and D-malate); Step 8Oxaloacetate is regenerated by the oxidation of L-malate; this reaction is catalyzed by malate dehydrogenase with two electrons collected by NAD+. The Citric Acid Cycle

29. 5. The citric acid cycle consists of eight successive reactions The aldol condensation between acetyl-CoA and oxaloacetate forms citrate The Citric Acid Cycle

30. 5. The citric acid cycle consists of eight successive reactions Citrate synthase before and after binding to oxaloacetate The Citric Acid Cycle Oxaloacetate Carboxylmethyl-CoA

31. 5. The citric acid cycle consists of eight successive reactions Citrate is converted to isocitrate via dehydration followed by a Hydration（水合作用） step. The Citric Acid Cycle 4Fe-4S cubic array:each Fe is bonded to three inorganic S and a cysteine sulfur atom (except one)

32. 5. The citric acid cycle consists of eight successive reactions The Citric Acid Cycle The first oxidation step Isocitrate is converted to a-ketoglutarate via an oxidative decarboxylation step, generating NADH CO2.

33. 5. The citric acid cycle consists of eight successive reactions TPP lipoate， FAD The Citric Acid Cycle (E1, E2, E3) The second oxidation step Thea-ketoglutarate dehydrogenase complex closely resembles the pyruvate dehyrogenase complex in structure and function

34. 5. The citric acid cycle consists of eight successive reactions Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation of ADP. The Citric Acid Cycle

35. 5. The citric acid cycle consists of eight successive reactions Succinyl-CoA Synthetase from E. coli The Citric Acid Cycle Coenzyme A His246-Pi The power helices

36. 5. The citric acid cycle consists of eight successive reactions The Citric Acid Cycle The third oxidation step (An enzyme bound to the inner membrane of mitochondria)

37. 5. The citric acid cycle consists of eight successive reactions The Citric Acid Cycle (a stereospecific enzyme)

38. 5. The citric acid cycle consists of eight successive reactions The Citric Acid Cycle (The fourth oxidation Step in the cycle) Oxaloacetate is regenerated at the end

39. 6. The complete oxidation of one glucose may yield as many as 32 ATP • All the NADH and FADH2 will eventually pass their electrons to O2 after being transferred through a series of electron carriers. • The complete oxidation of each NADH molecule leads to the generation of about 2.5 ATP, and FADH2 of about 1.5 ATP. • Overall efficiency of energy conservation is about 34% using the free energy changes under standard conditions and about 65% using actual free energy changes in cells. The Citric Acid Cycle

40. 6. The complete oxidation of one glucose may yield as many as 32 ATP The Citric Acid Cycle

41. 7. The citric acid intermediates are important sources for biosynthetic precursors • The citric acid cycle is the hub of intermediary metabolism serving both the catabolic分解代谢的and anabolic合成代谢的processes . • It provides precursors for the biosynthesis of glucose, amino acids, nucleotides, glucose, fatty acids, sterols, heme groups, etc. • Intermediates of the citric acid cycle get replenished （充满的）by anaplerotic（补缺的）reactions when consumed by biosynthesis. The Citric Acid Cycle

42. 8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently • The formation of acetyl-CoA from pyruvate is a key irreversible step in animals because they are unable to convert acetyl-CoA into glucose. • The complex (in all organisms) is allosterically inhibited by signaling molecules indicating a rich source of energy, e.g., ATP, acetyl-CoA, NADH, fatty acids; activated by molecules indicating a lack (or demand) of energy, e.g., AMP, CoA, NAD+, Ca2+. The Citric Acid Cycle

43. 8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently • The activity of the complex (in vertebrates, probably also in plants, but not in E. coli) is also regulated by reversible phosphorylation of one of the enzymes, E1, in the complex: phosphorylation of a specific Ser residue inhibits and dephosphorylation activates the complex. • The kinase and phosphatase is also part of the enzyme complex. • The kinase is activated by a high concentration of ATP. The Citric Acid Cycle

44. 8. The pyruvate dehydrogenase complex in vertebrates is regulated alloseterically and covalently The Citric Acid Cycle

45. 9. The rate of the citric acid cycle is controlled at three exergonic irreversible steps • Citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase; • Inhibited by product feedback (citrate, succinyl-CoA) and high energy charge (ATP, NADH); • Activated by a low energy charge (ADP) or a signal for energy requirement (Ca2+). The Citric Acid Cycle

46. 10. The partitioning of isocitrate, between the citric acid and glyoxylate乙醛酸cycles is coordinately regulated • The activity of the E. coli isocitrate dehydrogenase is inhibited when phosphorylated by a specific kinase and activated when dephosphorylated by a specific phosphatase. • The kinase and phosphatase activities are located in two domains of the same polypeptide and are reciprocally regulated: the kinase is allosterically inhibited (while the phosphatase activated) by molecules indicating an energy depletion, e.g., accumulation of intermediates of glycolysis and citric acid cycle. • The allosteric inhibitors of the kinase also act as inhibitors for the lyase: i.e., they activate the dehydrogenase while simultaneously inhibit the lyase. The Citric Acid Cycle

47. 10. The partitioning of isocitrate, between the citric acid and glyoxylate乙醛酸cycles is coordinately regulated The Citric Acid Cycle The isocitrate dehydrogenase and the isocitrate lyase are coordinately regulated.

48. Summary • Pyruvate is converted to acetyl-CoA by the action of pyruvate dehydrogenase complex, a huge enzyme complex. • Acetyl-CoA is converted to 2 CO2 via the eight-step citric acid cycle, generating three NADH, one FADH2, and one ATP (by substrate-level phophorylation). • Intermediates of citric acid cycle are drawn off to synthesize many other biomolecules, including fatty acids, steroids, amino acids, heme, pyrimidines, and glucose. The Citric Acid Cycle

49. Summary • Oxaloacetate can get supplemented from pyruvate, via a carboxylation reaction catalyzed by the biotin-containing pyruvate carboxylase羧化酶. • The activity of pyruvate dehydrogenase complex is regulated by allosteric effectors and reversible phosphorylations. • Net conversion of fatty acids to glucose can occur in germinating seeds, some invertebrates and some bacteria via the glycoxylate cycle, which shares three steps with the citric acid cycle but bypasses the two decarboxylation steps, converting two molecules of acetyl-CoA to one succinate. • Acetyl-CoA is partitioned into the glyoxylate乙醛酸cycle and citric acid cycle via a coordinately regulation of the isocitrate dehydrogenase and isocitrate lyase. The Citric Acid Cycle

50. References • De Kok, A., Hangeveld, A. F., Martin, A., and Westphal, A. H. (1998) “The pyruvate dehydrogenase multienzyme complex from gram-negative bacteria” Biochim. Biphys. Acta 1385:353-366. • Hagerhall, C. (1997) “Succinate:quinone oxidoreductase. Variations on a conserved theme” Biochim. Biophys. Acta 1320:107-141. • Knowles, J. (1989) “The mechanism of biotin-dependent enzymes” Annu. Rev. Biochem. 58:195-221. The Citric Acid Cycle