1. Chapter 20: The Calvin Cycle and the Pentose Phosphate Pathway Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition

2. Photosynthesis Dark Reactions (The Calvin cycle) Reductive conversion of CO 2 into carbohydrate. This process fixes ~10 10 tons of CO 2 annually. Process is powered by ATP and NADPH which are products of the light reactions of photosynthesis. Dark reactions occur in chloroplast stroma. Called the Calvin-Benson-Bassham pathway or the reductive pentosephosphate cycle (RPP)

3. Net equation for the Calvin cycle 3 CO 2 + 9 ATP + 6 NADPH + 5 H 2 O 9 ADP + 8 P i + 6 NADP + + *Triose phosphate From an energy standpoint this is an expensive process: 3 ATP and 2 NADPH per CO 2 incorporated. *(G3P or DHAP) These reactions occur in the stroma.

4. Calvin Cycle Fixation, Reduction, Regeneration

5. Stage 1: Incorporation of CO 2 Catalyzed by the enzyme Rubisco. Ribulose-1,5-bisphosphate carboxylase-oxygenase Plants fed CO 2, yield 3-phosphoglycerate as the first compound detected.

6. Rubisco 8 subunits @ 53000 8 subunits @ 14000 ~540000 d total It is the most abundant enzyme in the biosphere

7. Rubisco Mechanism

8. Mg ++ binding to Rubisco Bound using Glu, Asp and a Lys carbamate. The carbamate formation is catalyzed by Rubisco Activase

10. Stage 2 Phosphorylation and Reduction

11. Hexose synthesis 3-phospho glycerate to hexose-P

12. Stage 3: Regeneration These reactions serve to regenerate ribulose-1,5-bisphosphate from glyceraldehyde-3-phosphate. Two group transfer reactions are common here: 1. a transketolase reaction using TPP and 2. a transaldolase reaction Then an isomerase, an epimerase and a kinase complete the cycle.

13. Example Transketolase 2 C transfer from a ketose TPP

14. Example Transaldolase 3 C transfer from a ketose

15. Sugar Interconversions 2 C transfer from ketose TPP

16. Sugar Interconversions 3 C transfer from ketose

17. Sugar Interconversions 2 C transfer from ketose TPP

18. Sugar Interconversions

19. Calvin Cycle

20. Enzymes of the Calvin Cycle 1. Rubisco 2. Phosphoglycerate kinase 3. Glyceraldehyde-3-phosphate dehydrogenase 4. Triosephosphate isomerase 5. Aldolase (transaldolase) 6. Fructose bisphosphatase 7. Transketolase 8. Sedoheptulose-7-phosphatase 9. Phosphopentose isomerase 10. Phosphopentose epimerase 11. Ribulose-5-phosphate kinase

21. Carbon Flow in the Calvin Cycle 3 C 5 + 3 C 1 ---> 6 C 3 2 C 3 ---> 1 C 6 C 6 + C 3 ---> C 4 + C 5 C 4 + C 3 ---> C 7 C 7 + C 3 ---> 2 C 5 ----------------------------------- net 3 C 1 ---> 1 C 3

22. Synthesis of Sucrose Occurs in the cytosol. There is a triose-P:Pi antiport in the chloroplast membrane.

23. Regulation of the Calvin Cycle Rubisco has three forms: 1.E binds R-1,5-B in the dark and is inactive. R-1,5-B is an inhibitor in the dark (blocks the carbamylation site). Rubisco activase causes dissociation of R-1,5-B and catalyzes ATP dependent attachment of CO 2. 2.EC carbamylated at Lys 201 is still inactive. 3.ECM has bound Mg ++ and is active. Rubisco and rubisco activase are light activated.

24. Regulation of the Calvin Cycle Rubisco activation requires: light, CO 2, Mg ++ & pH 7.4 (optimum is 8.1) Functioning PSII-Cytbf-PSI cause proton pumping which leaves the stroma basic. The potential developed promotes translocation of Mg ++ and Cl -. Mg ++ is needed in the stroma for rubisco and both phosphatases. The stroma can reach pH 9.0. The high pH activates rubisco. Light produces a conformational change in rubisco activase enhancing its activity.

25. Regulation of the Calvin Cycle Functioning PSII-Cytbf-PSI also produces reduced ferredoxin and NADPH. Ferredoxin-Thioredoxin Reductase generates thioredoxin-(SH) 2 Ferredoxin(red) + thioredoxin-S 2 (ox) ===== > Ferredoxin(ox) + thioredoxin-(SH) 2 (red) Thioredioxin (a small protein) activates a number of enzymes through a disulfide -- > dithiol conversion.

26. Regulation of Photosynthesis Light is needed to generate NADPH, FD red and Mg ++ transport

27. Thioredoxin This is a small disulfide containing protein.

28. Action of Thioredoxin Enzymes activated by thioredoxin: Fructose bisphosphatase Sedoheptulose bisphosphatase Ribulose-5-P kinase Glyceraldehyde-3-P dehydrogenase

29. Regulation

30. Photorespiration (Use of O 2 ) This competes with photosynthesis at higher temperatures so is typically more active in the summer and in the tropics. Conc. in air K M O 2 250 μM (20%) 200 μM CO 2 11 μM (0.04%) 20 μM However, the affinity of Rubisco for CO 2 decreases with increasing temperature. The immediate products of photorespiration are phosphoglycolate and 3-phosphoglycerate.

31. Photorespiration The mechanism is analogous to that for carboxylation. Rubisco must be carbamylated.

32. Photorespiration The reaction involved in photorespiration require participation of enzymes from the cytosol, chloroplasts, mitochondria and peroxisomes. Chloroplast:glycolate phosphatase Peroxisome:glycolate oxidase transaminase hydroxypyruvate reductase Mitochondria:Glycine cleavage enzyme serine hydroxymethyltransferase Cytosol:glycerate kinase

33. Photo respiration reactions

35. Photorespiration

36. Run through the previous reactions twice yield two glycines which then react as shown below. So two glycolates produce 1 CO 2 and 1 serine.

37. Tetrahydrofolate FH is a coenzyme that serves as a one carbon carrier.

38. Hatch-Slack Pathway This pathway uses a CO 2 concentrating mechanism to permit photosynthesis to surpass photorespiration. The initially observed compound is this case is oxaloacetate, so this is referred to as the C4 pathway. Similarly, normal photosynthesis is sometimes called the C3 pathway. C4 plants include crabgrass, bermuda grass, corn, maize and sugarcane. These have an advantage over C3 plants in hot weather.

39. C 4 Pathway C4 plants have mesophyll cells which are outer cells that collect CO 2. Bundle sheath cells are inside where the Calvin Cycle occurs.

40. C 4 Pathway

41. Hatch-Slack Pathway Different plants have different mechanisms for moving CO 2 into the bundle sheath cells. The Hatch- Slack uses four enzymes in the C4 pathway. 1. PEP carboxylase: HOH + PEP + CO 2 -- > OAA + Pi 2. Malate dehydrogenase (NADP + dependent) 3. Malic enzyme (NADP + dependent) also called malate dehydrogenase decarboxylating 4. Pyruvate:phosphate dikinase ATP + Pi -- > ADP + PPi ADP + E -- > AMP + E~P E~P + Pyruvate -- > PEP + E PPi -- > 2 Pi

42. Hexose Monophosphate Shunt (HMS) Pentose phosphate pathway or Phosphogluconate pathway This pathway is the major site for production of: 1. NADPH for anabolism (reductive) 2. Ribose-5-phosphate for nucleotide synth. Other: Makes erythrose for Phe synthesis. Completely oxidizes glucose without Krebs. No ATP used or made in this pathway.

44. Hexose monophosphate shunt (HMS) Phase 1 - oxidative. Phase 2 – isomerization, epimerization and rearrangement.

45. HMS This phase is composed of three reactions, two of which are oxidations.

46. HMS Isomerization and epimerizartion.

47. HMS

48. HMS – Oxidative Phase The oxidative phase is one-way as written. Reaction 1 is the control site for the HMS. Reaction 3 is the least reversible step. The mechanism involves the decarboxylation of a  -ketoacid, similar to isocitrate dehydrogenase.

49. HMS – 1st Oxidative Step

50. HMS – Lactone Formation

51. HMS – 2nd Oxidative Step

52. Sugar Interconversions 2 C transfer from ketose TPP

53. Sugar Interconversions 3 C transfer from ketose

54. Sugar Interconversions 2 C transfer from ketose TPP

55. Pentose Phosphate Pathway Reactions

56. Transketolase Mechanism

57. Step 1

58. Step 2

59. Step 3

60. Step 4

61. Step 5

62. Transaldolase Mechanism

63. Step 1

64. Step 2

65. Step 3

66. Step 4

67. Step 5

68. Step 6

69. Transaldolase & Transaldolase Active components of each mechanism transketolase transaldolase

70. Making Ribose-5-P Enter HMS from F-6-P and convert all to ribose-5-P

71. Making NADPH & Ribose-5-P Enter HMS from G-6-P, make NADPH and convert all ribulose-5-P to ribose-5-P

72. Making NADPH Enter HMS from G-6-P, convert all ribulose-5-P to fructose-6-P and recycle to glucose-6-P.

73. Making NADPH & ATP Enter HMS from G-6-P, convert all ribulose-5-P to fructose-6-P and use it for energy via glycolysis.

74. Active HMS

75. Regulation of the HMS Glucose-6-phosphate dehydrogenase is the control point for the pathway. NADPH(-) competes for the binding site with NADP +. Also, fattyacyl CoA(-). Thus, regulation is tied to the need for anabolism and reductive processes. K M of the enzyme for NAD + is 1000 times greater that that for NADP +. Normal levels: NAD + /NADH = 700-1000 and a high level of NAD favors oxidation reactions. NADP + /NADPH = 0.01-0.014 and high levels of NADP favor reduction reactions.

76. Erythrocytes The need for NADPH in red cells is critical. Red cells lack mitochondria therefore no energy is available from the Krebs cycle or ET/OP. All energy is derived from glycolysis and the HMS. NADPH is needed to keep hemoglobin and other proteins in the active dithiol form. The active agent here is glutathione, a ubiquitous reducing agent (found in all cells). Glutathione is  -glutamyl-cysteinylglycine (GSH). Activation of an oxidized enzyme: 2 GSH + ES 2 -- > GSSG + E(SH) 2

77. Glutathione (Found in all cells.)  - carboxyl thiol

78. Reduction of GSSG Glutathione reductase is an NADPH requiring flavoprotein that catalyzes conversion of GSSG back to 2 GSH. FADH 2 does not convert GSSG directly but rather goes through a disulfide/ dithiol conversion on the enzyme. NADPH + H + + E-FAD -- > NADP + + E-FADH 2 E-FADH 2 + ES 2 -- > E-FAD + E(SH) 2 E(SH) 2 + GSSG -- > ES 2 + 2 GSH The normal GSH/GSSG ratio in red cells is ~500.

79. End of Chapter 20 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition