1. The Calvin Cycle and the Pentose Phosphate Pathway
Chapter 20
The Calvin Cycle and the Pentose Phosphate Pathway
Atmospheric carbon dioxide measurements at Mauna Loa, Hawaii. These measurements show annual cycles resulting from seasonal variation in carbon dioxide fixation takes place in rain forests, which account for approximately 50% of terrestrial fixation.

2. Outline
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
20.2. The Activity of the Calvin Cycle Depends on Environmental Conditions
20.3 The Pentose Phosphate Pathway Generates NADPH and Synthesizes Five-Carbon Sugars
20.4. The Metabolism of Glucose 6-Phosphate by the Pentose Phosphate Pathway Is Coordinated with Glycolysis
20.5. Glucose 6-Phosphate Dehydrogenase Plays a Key Role in Protection Against Reactive Oxygen Species

3. General Features of photosynthesis
Photosynthesis in plants encompasses two processes:
the light-dependent reactions, or light reactions
Transform light energy into ATP
Biosynthetic reducing power, NADPH
dark reactions (Calvin cycle or carbon-fixation reactions
Does nor directly depend on the light
Use the ATP and NADPH to reduced carbon atoms
CO2 or hexose
The light reactions and dark reactions of photosynthesis cooperate to transform light energy into carbon fuel
p.742

4. Pentose phosphate pathway 五碳糖磷酸途徑
Common to all organisms
Known variously as the pentose phosphate pathway, the hexose monophosphate pathway, the phosphogluconate pathway or pentose shunt
Break down glucose into carbon dioxide to generate NADPH
Glycolysis  Gluconeogenesis
Calvin cycle  Pentose phosphate pathway

5. Compare Cavin cycle with pentose phosphate pathway
dark reaction
hexose monophosphate pathway
the phosphogluconate pathway
pentose shunt
Uses NADPH to reduce carbon dioxide to generate hexoses
Breaks down glucose into carbon dioxide to generate NADPH
Reductive pentose phosphate pathway
Oxidative pentose phosphate pathway
catabolism of pentose sugars from the diet
the synthesis of pentose sugars for nucleotide biosynthesis
catabolism and synthesis of less common four- and seven-carbon sugars

6. 20.1 The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Photosynthetic organism can use the Calvin Cycle to synthesize glucose from CO2 and water, by using sunlight as an energy source
takes place in the stroma of chloroplasts
The Calvin cycle comprises three stages:
Stage 1:
The fixation of CO2 by ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate.
Stage 2:
The reduction of 3-phosphoglycerate to form triose (hexose) sugars.
Stage 3:
The regeneration of ribulose 1,5-bisphosphate so that more CO2 can be fixed

7. The Calvin cycle comprises three stages
DHAP

8. Fixation of CO2-- Carbon dioxide reacts with Ribulose 1,5-bisphosphate to form two 3-phosphoglycerate
Highly exergonic reaction (ΔG0’=-51.9kJ/mol)
Catalyzed by Ribulose 1,5-bisphosphate carboxylase/ oxygenase (rubisco)
A soluble enzyme located on the stromal surface of the thylakoid membrane of chloroplasts
Unstable intermediate

9. Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco)
Very abundant in chloroplasts, constituting more than 30% of the total leaf protein in some plants
Large amounts are present because rubisco is a slow enzyme; its maximal catalytic rate is only 3s-1.
Unique to the Cavin cycle
The rate-limiting step in hexose synthesis
consists of 8 large (L, 55kd) subunits and 8 small (S, 13kd) subunits.
Each L chain contains a catalytic site and a regulatory site
The S chains enhance the catalytic activity of the L chains

10. Biochemistry Rubisco activity depends on magnesium ion and carbamate
Rubisco requires a bound divalent metal ion for activity, usually magnesium ion
activate a bound substrate molecule by stabilizing a negative charge
CO2 is required to complete the assembly of the Mg2+ binding site in rubisco
CO2 molecule adds to the uncharged ε-amino group of lysine 201 to form a carbamate
is facilitated by the enzyme rubisco activase
Metal center
Berg • Tymoczko • Stryer
rubisco activase
Biochemistry
Seventh Edition
CHAPTER 20
The Calvin Cycle and the Pentose Phosphate Pathway
Copyright © 2012 by W. H. Freeman and Company

11. Role of the Magnesium Ion in the Rubisco Mechanism
Ribulose 1,5-bisphosphate binds to a magnesium ion that is linked to rubisco through a glutamate residue, an aspartate residue, and the lysine carbamate
Ribulose 1,5-bisphosphate binds to Mg2+ through its keto group and an adjacent hydroxyl group
The coordinated ribulose 1,5-bisphosphate gives up a proton to form a reactive enediolate species that reacts with CO2 to form a new carbon-carbon bond

12. Overall pathway for the conversion of ribulose 1,5 bisphosphate
and CO2 into two molecules of 3-phosphoglycerate H+

13. Overall pathway for the conversion of ribulose 1,5 bisphosphate
and CO2 into two molecules of 3-phosphoglycerate

14. Overall pathway for the conversion of ribulose 1,5 bisphosphate
and CO2 into two molecules of 3-phosphoglycerate
Stage 1:
The fixation of CO2 by ribulose 1,5- bisphosphate to form two molecules of 3-phosphoglycerate.

15. Rubisco also catalyzes a wasteful oxygenase reaction
Calvin cycle
Rubisco also catalyzes a wasteful oxygenase reaction
Photorespiratory
The rate of carboxylase reaction is four times than the oxygenase reaction
The oxygenase reaction requires the carbamate forms
Carbamate forms only in the presence of CO2
When CO2 is absent  No oxygenase reaction

16. (C2 oxidative photosynthetic carbon cycle)
Photorespiration
(C2 oxidative photosynthetic carbon cycle)
Because O2 is consumed and CO2 is released
No ATP and NADPH or another energy produced
Loss of up 25% of the carbon fixed
phosphatase
Glyoxylate oxidase
With FMN
catalase
H2O and O2
transamination
Fig 20.6 photorespiratory reactions

17. Glycolate pathway. Part I

18. Glycolate pathway. Part II

19. Glycolate pathway. Part III
Chloroplast , glutamine synthesis
p.787

20. Glycolate pathway. Part VI

21. Glycolate pathway. Part V

23. Exported to the cytosol Phosphoglycerate kinase
Hexose phosphates are made from phosphoglycerate, and ribulose 1,5-phosphate is regenerated
These reactions and that catalyzed by rubisco bring CO2 to the level of a hexose, converting CO2 into a chemical fuel at the expense of NADPH and ATP generated from the light reactions
The 3-phosphoglycerate product of rubisco is next converted into three forms of hexose phosphate: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate: hexose monophosphate pool
Starch is synthesized and stored in chloroplasts
Regeneration of RuBP
(GAP)
Triose-phosphate
isomerase
Glyceraldehyde
3-phosphate
dehydrogenase
Exported to the cytosol
Phosphoglycerate kinase
Sucrose synthesis
(3PGA)
Fig 20.8 Hexose phosphate formation

24. The Calvin cycle comprises three stages
Fixation
(3C)
(7C)
Stage 3
regeneration
(7C)
(3C)
(5C)
(4C)
(3C)
(6C)
Stage 2
reduction
(6C)
(3C)
(3C)

25. How to construct a five-acrbon sugar from six-carbon sugar??
The reaction catalyzed by the transketolase and aldolase
Transketolase
requires the coenzyme thiamine pyrophosphate (TPP) to transfer a two-carbon unit (CO-CH2OH) from a ketose to an aldose

26. Aldolase
catalyzes an aldol condensation between dihydroxyacetone phosphate (DHAP) and an aldehyde.
This enzyme is highly specific for dihydroxyacetone phosphate, but it accepts a wide variety of aldehydes
Donor : keto, 3C
Acceptor : Aldose

27. Stage 3:
The regeneration of ribulose 1,5-bisphosphate so that more CO2 can be fixed

28. Unique to the Cavin cycle

29. (Unique to the Calvin cycle)
Epimer
-Regeneration of Ribulose 1,5-Bisphosphate. Both ribose 5-phosphate and xylulose 5-phosphate are converted into ribulose 5-phosphate, which is then phosphorylated to complete the regeneration of ribulose 1,5-bisphosphate.

30. Stoichiometry of CO2 assimilation in the Calvin cycle
p.782

31. Three ATP and two NADPH molecules are used to bring carbon dioxide to the level of a hexose
6 CO ATP + 12 NADPH + 12 H2O
C6H12O ADP + 18 Pi + 12 NADP+ + 6H+
12 ATP are expended in phosphorylating 12 molecules of 3-phosphoglycerate to 1,3-bisphosphoglycerate
12 NADPH are consumed in reducing 12 molecules of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate
An additional six molecules of ATP are spent in regenerating ribulose 1,5-bisphosphate
The outcome of the Calvin cycle is the generation of a hexose and the regeneration of the starting compound, ribulose 1,5-bisphosphate

32. Starch and sucrose are the major carbohydrate stores in plants
Starch is synthesized and stored in chloroplasts
Assimilation of CO2 into biomass in plants.

33. A Transport System Exports Triose Phosphates from the Chloroplast and Imports Phosphate
The inner chloroplast membrane is impermeable to most phosphorylated compounds
a specific antiporter that catalyzes the one-for-one exchange of Pi with a triose phosphate, either dihydroxyacetone phosphate or 3-phosphoglycerate
The Pi–triose phosphate antiport system has the indirect effect of moving ATP equivalents and reducing equivalents from the chloroplast to the cytosol
p.783

34. Synthesis of sucrose Sucrose + Pi
Sucrose, a disaccharide, is synthesized in the cytoplasm
Plant lack the ability to transport hexose phosphates across the chloroplast membrane
they are able to transport triose phosphates from chloroplasts to the cytoplasm
Sucrose phosphate phosphatase
Sucrose + Pi

35. Important!!!!

36. The Activity of the Calvin Cycle Depends on Environmental Conditions
The principal means of regulation is alteration of the stromal environment by the light reactions
Light reactions increase in stromal pH and in the concentrations of Mg2+, NADPH and reduced ferredoxin
all of which contribute to the activation of Calvin-cycle enzymes
lumen
Stroma H+
e-, Mg2+

37. Rubisco is activated by light-driven changes in stromal pH and [Mg2+]
Light facilitates the carbamate formation necessary to enzyme activity
The activity of rubisco increases markedly on illumination
the light-driven pumping protons into the thylakoid space
In the stroma, the pH increases from 7 to 8 , and the level of Mg2+ rises
Carbamate formation is favored by alkaline pH.
Light leads to generation of regulatory signal as well as ATP and NADPH

38. Thioredoxin plays a key role in regulating the Calvin cycle
a 12-kd protein containing neighboring cysteine residues that cycle between a reduced sulfhydryl and an oxidized disulfide form
The reduced form of thioredoxin activates many biosynthetic enzymes by reducing disulfide bridges
In the chloroplasts
Oxidized thioredoxin is reduced by ferredoxin by ferredooxin-thioredoxin reductase
4Fe-4S cluster
Couples two one-electron oxidation of reduced ferredoxin to the two-electron reduction of thioredoxin
NADPH is a signal molecule
Activates phosphoribulose kinase and glyceraldehyde 3-phsophate dehydrogenase
In the dark, these enzymes are inhibited by associated with an 8.5kd CP12
NADPH disrupts this association
red ox

39. Enzymes regulated by thioredoxin
(via Rubisco activase)

40. The C4 Pathway of Tropical Plants Accelerates Photosynthesis by Concentrating Carbon Dioxide
At higher temperatures, the ratio of photorespiration to photosynthesis increases (oxygenase activity increase more rapidly with temperature than does the carboxylase activity)
Using a high local concentration of CO2 at the site of the Calvin cycle in their photosynthetic cells.
Four-carbon (C4) compounds such as oxaloacetate and malate carry CO2 from mesophyll cells (contact with air), to bundle-sheath cells (維管束鞘細胞) ( the major sites of photosynthesis)
Decarboxylation of the four-carbon compound in a bundle- sheath cell maintains a high concentration of CO2 at the site of the Calvin cycle
The three-carbon compound pyruvate returns to the mesophyll cell for another round of carboxylation.

41. CO2 (in mesophyll cell) + ATP +2H2O 
CO2 (in bundle-sheath cell) + AMP + 2Pi + 2H+
The CO2 concentration can be 20 fold as great in the bundle sheath cells as in the mesophyll cells
NADP+ -linked malate dehydrogenase
NADP+ -linked malic enzyme
PEP carboxylase
pyruvate,Pi dikinase

42. When the C4 pathway and the Calvin cycle operate together, the net reaction is24
Tropical plants with a C4 pathway do little photorespiration because the high concentration of CO2 in their bundle sheath cells accelerates the carboxylase reaction relative to the oxygenase reaction
C4 plants have the advantage in a hot environment and under high
illumination, which accounts for their prevalence in the tropics
C3 plants, which consume only 18 molecules of ATP per hexose molecule formed in the absence of photorespiration (compared with 30 molecules of ATP for C4 plants), are more efficient at temperatures of less than about 28°C, and so they predominate in temperate environments

43. Crassulacean (景天科) Acid Metabolism (CAM) Permits Growth in Arid Ecosystems
open
In Hot, dry climates
The stomata is closed to prevent water loss
CO2 cannot be absorbed during daylight hours
When the stomata open at the cooler temperatures of night, CO2 is fixed by the C4 pathway into malate, which is stored in vacuoles. During the day, malate is decarboxylated and the CO2 becomes available to the Calvin cycle
In contrast with C4 plants, CAM plants separate CO2 accumulation from CO2 utilization temporally rather than spatially

44. The Pentose Phosphate Pathway Generates NADPH and Synthesizes Five-Carbon Sugars
Photosynthetic organisms can use the light reactions for generation of some NADPH
In all organisms, the Pentose Phosphate Pathway generates NADPH and synthesizes five-carbon sugars
takes place in the cytosol
is a crucial source of NADPH to use in reductive biosynthesis as well as for protection against oxidative stress

45. Pentose Phosphate Pathway
It consists of 2 phases:
– Oxidative generation of NADPH
– Nonoxidative interconversion of sugars

46. Phase 1: Oxidative generation of NADPH
NADPH is generated when glucose 6-phosphate is oxidized to ribulose 5-phosphate, which is subsequently converted into ribose 5-phosphate.
Ribose 5-phosphate and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A

47. two Molecules of NADPH Are Generated in the Conversion of Glucose 6-phosphate into Ribulose 5-phosphate
Hydrolysis
Highly specific to NADP+
Oxidatively decarboxylation

48. 3. 6-Phosphogluconate Dehydrogenase
-Oxidatively decarboxylation of 6-phosphogluconate to ribulose 5-
phosphate. NADP+ is again the electron acceptor

49. Nonoxidative interconversion of sugars
The non-oxidative phase of the pentose phosphate pathway provides a link to glycolysis
By Transketolase and Transaldolase

50. C5 C5
ketose
aldose C5 C5

51. Transaldolase
ribose 5-phosphate is converted into the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate by transketolase and transaldolase
These enzymes create a reversible link between the pentose phosphate pathway and glycolysis by catalyzing these three successive reactions
Net reaction :

52. The first of the three reactions linking the pentose phosphate pathway and glycolysis is the formation of glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate from two pentoses

53. Transaldolase

55. The sum of these reactions is :
Excess ribose 5-phosphate formed by the pentose phosphate pathway can be completely converted into glycolytic intermediates. Moreover, any ribose ingested in the diet can be processed into glycolytic intermediates by this pathway

57. Transketolase and Transaldolase
Transketolase transfers a two-carbon unit, whereas transaldolase transfers a three-carbon unit
From a ketose donor to an aldose acceptor

58. Transketolase Mechanism
(1) Thiamine pyrophosphate (TPP) ionizes to form a carbanion (2)The carbanion of TPP attacks the ketose substrate. (3) Cleavage of a carbon-carbon bond frees the aldose product and leaves a two-carbon fragment joined to TPP. (4) This activated glycoaldehyde intermediate attacks the aldose substrate to form a new carbon-carbon bond. (5) The ketose product is released, freeing the TPP for the next reaction cycle

59. Transaldolase Mechanism
leaving a three-carbon fragment attached to the lysine
(1) The reaction begins with the formation of a Schiff base between a lysine residue in transaldolase and the ketose substrate. Protonation of the Schiff base (2) leads to release of the aldose product, leaving a three-carbon fragment attached to the lysine residue. (4) This intermediate adds to the aldose substrate to form a new carbon-carbon bond. Subsequent deprotonation (5) and hydrolysis of the Schiff base (6) release the ketose product from the lysine side chain, completing the reaction cycle.

60. Fig 20.22 Carbanion intermediates

61. The Metabolism of Glucose 6-Phosphate by the Pentose Phosphate Pathway Is Coordinated with Glycolysis
Glucose 6-phosphate is metabolized by both the glycolytic pathway and the pentose phosphate pathway
How is the processing of this important metabolite partitioned between these two metabolic routes ?
The cytoplasmic concentration of NADP+ plays a key role in determining the fate of glucose 6-phosphate

62. The rate of the pentose phosphate pathway is controlled by the level of NAPD+
Oxidative Phase
Dehydrogenation f G6P: irreversible reaction
control site (rate-limiting step)
Regulated by ratio of NADP+/ NADPH levels
low level of NADP+, reduce the dehydrogenation of glucose 6-phosphate
NADP+ is needed as the electron acceptor
NADPH competes with NADP+ in binding to the enzyme
ensures that NADPH is not generated unless the supply needed for reductive biosyntheses is low
Nonoxidative Phase
Controlled by availability substrates

63. Interplay between glycolysis and the pentose phosphate pathway by examining the metabolism of glucose 6-phosphate

64. Mode 1: Much more ribose 5-phosphate than NADPH is required
Rapidly dividing cells need ribose 5- phosphate for the synthesis of nucleotide precursors of DNA
Most of the glucose 6-phosphate is converted into fructose 6-phosphate and glyceraldehyde 3- phosphate by the glycolytic pathway
Transaldolase and transketolase then convert two fructose 6-phosphate and one glyceraldehyde 3-phosphate into three ribose 5-phosphate
glycolytic pathway
Transaldolase and transketolase
3 Ribose 5-phosphate ↔ 2 fructose 6-phosphate + glyceraldehyde 3-phosphate
Stoichiometry of mode 1 :
5 Glucose 6-phosphate + ATP →
6 ribose 5-phosphate + ADP + 2H+

65. Phosphopentose isomerase
Mode 2: The needs for NADPH and ribose 5-
phosphate are balanced
Formation of two molecules of NADPH and one molecule of ribose 5-phosphate from one molecule of glucose 6-phosphate in the oxidative phase of the pentose phosphate pathway
Phosphopentose isomerase
Stoichiometry of mode 2 :
Glucose 6-phosphate + 2 NADP+ + H2O →
ribose 5-phosphate + 2 NADPH + 2 H+ + CO2

66. Mode 3: Much more NADPH than ribose 5-phosphate is required
Adipose tissue requires a high level of NADPH for the synthesis of fatty acids
G6P is completely oxidized to CO2

67. Mode 3: Much more NADPH than ribose 5-phosphate is required
Oxidative phase of the pentose phosphate pathway:
6 Glucose 6-phosphate + 12 NADP+ + 6H2O →
6 ribose 5-phosphate + 12 NADPH + 12 H+ + 6CO2
Transketolase and transaldolase
6 Ribose 5-phosphate ↔
4 fructose 6-phosphate + 2 glyceraldehyde 3-phosphate
Gluconeogenic pathway
4 Fructose 6-phosphate + 2 glyceraldehyde 3-phosphate +H2O↔ 5 glucose 6-phosphate +Pi
G6P is completely oxidized to CO2 with the generation of NAPDH

68. The sum of the mode 3 reactions is
Glucose 6-phosphate + 12 NADP+ + 7 H2O →
6 CO NADPH + 12 H+ + Pi
The equivalent of glucose 6-phosphate can be completely oxidized to CO2 with the concomitant generation of NADPH
Ribose 5-phosphate produced by the pentose phosphate pathway is recycled into glucose 6-phosphate by transketolase, transaldolase, and some of the enzymes of the gluconeogenic pathway

69. Mode 4: Both NADPH and ATP are required
Ribose 5-phosphate formed by the pentose phosphate pathway can be converted into pyruvate
Fructose 6-phosphate and glyceraldehyde 3-phosphate derived from ribose 5-phosphate enter the glycolytic pathway
ATP and NADPH are concomitantly generated
TCA cycle
3 Glucose 6-phosphate + 6 NADP+ + 5 NAD++ 5 Pi + 8 ADP →
5 pyruvate + 3 CO2 + 6 NADPH + 5 NADH+ 8 ATP + 2 H2O + 8 H+

70. Glucose 6-Phosphate Dehydrogenase Plays a Key Role in Protection Against Reactive Oxygen Species
The NADPH generated by the pentose phosphate pathway plays a vital role in protecting the cells from reactive oxygen species (ROS)
Reactive oxygen species (ROS)– Chemically reactive molecules containing oxygen, e.g., hydrogen peroxides (H2O2)
Reduction of O2 :
O2 → O2·- → O22-
Reactive oxygen species (ROS) generated in oxidative metabolism inflict damage on all classes of macromolecules and can ultimately lead to cell death
e -
Superoxide
ion
Peroxide

71. Reduced glutathione (GSH), a tripeptide with a free sulfhydryl group, combats oxidative stress by reducing ROS to harmless forms
The glutathione in the oxidative form (GSSG) and must be reduced to generate GSH
The reducing power is supplied by the NADPH generated by glucose 6- phosphate dehydrogenase in the pentose phosphate pathway

72. Compare Cavin cycle with pentose phosphate pathway
dark reaction
hexose monophosphate pathway
the phosphogluconate pathway
pentose shunt
Uses NADPH to reduce carbon dioxide to generate hexoses
Breaks down glucose into carbon dioxide to generate NADPH
Reductive pentose phosphate pathway
Oxidative pentose phosphate pathway
catabolism of pentose sugars from the diet
the synthesis of pentose sugars for nucleotide biosynthesis
catabolism and synthesis of less common four- and seven-carbon sugars