1. Calvin Cycle & Pentose Phosphate Pathway

2. Light reactions of Photosynthesis generate ATP and NADPH
At thylakoid membrane in plants
The ATP and NADPH are used to fix carbon dioxide in the dark reactions.

3. The Calvin Cycle fixes CO2 into sugars

4. Rubisco catalyzes the carboxylation of RBP
CO2 is condensed with ribulose-1,5-biphosphate to form an unstable intermediate that hydrolyzes to two 3-Phosphoglycerates
Catalyzed by ribulose 1,5-bisophosphate carboxylase/oxygenase (Rubisco).
Highly exergonic: DG0′ = kJ mol-1.
Rate limiting step in hexose synthesis.

5. RUBISCO Rubisco is the most abundant enzyme on earth.
Found on the stromal surface of thylakoid membranes in chloroplasts.
Has 8 large subunits (55 kDa, L) & 8 small subunits (13 kDa, S).
Each L chain has a catalytic site and a regulatory site.
Slow: maximal rate 3 s-1.
A second CO2 must be bound to a lysine for the enzyme to be active.

6. Rubisco Structure
CO2 is bound to bind critical Mg2+

7. Rubisco Carboxylation on Mg2+

8. Rubisco also catalyzes oxidation of Ribulose-1,6-bisphosphate
Oxidation occurs at 25% the rate of carboxylation at normal atmospheric conditions.
10 mM CO2, 250 mM O2
Wastes Ribulose without fixing carbon.
Requires the CO2 bound to lysine, so is prevented when CO2 is low.
Oxidation increases with temperature.

9. Recovery of Glycolate carbon
3 out of 4 carbons are recovered from 2 glycolate molecules via conversion to Gly and Ser.
Process is called photorespiration, since O2 is used and CO2 released.

10. Production of hexose phosphates
Process of Fructose and Glucose phosphate generation from 3-phosphoglycerate is similar to Gluconeogenesis.
GAP dehydrogenase in chloroplast uses NADPH instead of NADH.
3-phosphoglycerate can be moved to the cytoplasm and used for gluconeogenesis.
Glc-1-P, Glc-6-P, and Frc-6-P are interconvertable.

11. The third phase is to regenerate Ribulose-1,5-Bisphosphate.
Use transketolase and aldolase to convert 3 and 6 carbon sugars to a 5 carbon sugar.
Convert to Ribose and Xylulose 5-phosphates first.

12. Ribulose-1,6-Phosphate Production
What is wrong with the sugar structure on this slide?
Ribose 5-P and Xylulose 5-P are readily isomerized and epimerized Ribulose-5-P.
Ribulose-5-P is phosphorylated to R-1,5-BP.

13. Summary of Calvin Cycle
To make one hexose:

14. Sugars are stored as Starch & Sucrose
Starch is built up from ADP-Glucose, similar to Glycogen from UDP-Glucose
Sucrose-6-P is made from Fructose-6-P and UDP-Glucose, then dephosphorylated.

15. Pentose Phosphate Pathway
Synthesis of 5 Carbon Sugars and NADPH in nonphotosynthetic organisms.

16. Requirements for NADPH
Biosynthetic pathways & Detoxification require NADPH as the reducing agent.
Pathways requiring NADPH:
Fatty acid biosynthesis
Cholesterol biosynthesis
Neurotransmitter biosynthesis
Nucleotide biosynthesis
Reduction of oxidized glutathione (detoxification)
Cytochrome P450 monooxygenases (detoxification)

17. Tissues with Active Pentose Phosphate Pathway
Function
Adrenal gland
Steroid synthesis
Liver
Fatty acid and cholesterol synthesis
Testes
Adipose tissue
Fatty acid synthesis
Ovary
Mammary gland
Red blood cells
Maintenance of reduced glutathione
Berg, Tymoczko, Stryer, 5th Ed., Table 20.4
Copyright © 2002, W. H. Freeman and Company

18. Phases of Pentose Phosphate Pathway
The first phase of the PPP is oxidative decarboxylation to generate NADPH.
Gives 5-carbon sugars for nucleotide synthesis.
Sugars are rearranged in Phase 2.

19. Oxidative Phase of PPP Two molecules of NADP are reduced in 3 steps.
The ribulose-5-P can then be isomerized to ribose-5-P by phophopentose isomerase.

20. Stage 2: Rearrangement of sugars
Transaldolase and transketolase rearrange sugars to link the PPP to glycolysis.
Rearrangements similar to the Calvin Cycle.
Reactions are reversible, so sugars can be moved between the two pathways.
If the main purpose is to make NADPH, the reactions can be summarized as:
Net reaction:
Transaldolase

21. PPP rearrangement reactions
Rxn 1
Rxn 2
Rxn 3

22. Effect of PPP Second Stage
Or, including conversion of ribose-5-P:
Excess ribose-5-P produced in generating NADPH can be converted to glycolysis or glycolytic pathway intermediates.
Ribose from diet can be used in glycolysis or gluconeogenesis.
Ribose-5-P can be generated for nucleotide synthesis.

23. Four possible modes of using PPP

24. Glutathione is reduced by NADPH
Glutathione maintains a reducing environment in the cytoplasm & protects against reactive oxygen species (ROS).
Glutathione reductase uses NADPH to reduce oxidized glutathione (GSSG) to the reduced form (GSH).
NADPH is generated by Glucose-6-P-dehydrogenase (G6PD) & 6-Phosphoglucanate dehydrogenase of the PPP & malic enzyme in the cytosol/mitochondrial acetylCoA shuttle.
RBC’s that have no mitochondria are susceptible to oxidative damage in the case of G6PD deficiency.
This is common in Thailand and other areas where malaria has been prevalent.
The oxidizing environment & lack of PPP products in the RBC leads to poor growth of Plasmodium species.

25. Summary
The Calvin cycle uses NADPH and ATP to fix CO2 into six carbon sugars, while the pentose phosphate pathway (PPP) uses release of CO2 from a six carbon sugar to generate NADPH.
The Calvin cycle rearranges 6 and 3 carbon sugars to regenerate the 5-carbon starting material (ribulose-5-phosphate → ribulose-1,5-bisphosphate), while the PPP rearranges the 5 carbon product to 3 and 6 carbon sugars for use in glycolysis and glucose storage.
Most steps are reversible, so the same enzymes can be used in either direction for many of the PPP and Calvin cycle steps.
The directions and products of the pathways are detemined by cellular needs.

26. Glycogen & Starch metabolism
Glucose storage

27. Glycogen metabolism
Glycogen is a common and efficient storage form of glucose
Glycogen is synthesized and broken down by different pathways.
Synthesized by Glc-6-P > Glc-1-P>UDP-Glc > Glycogen
Broken down by phosphorylase: Glycogen + Pi > Glc-1-P > Glc-6-P
Glycogen synthesis and degradation are reciprocally regulated.
Synthesize when fuel levels are high.
Breakdown when Glucose (liver/blood) or energy (muscles) is needed.

28. Glycogen Breakdown
Glycogen 1,4-Glc are removed by glycogen phosphorylase.
Glycogen is debranched near 1,6 branchpoint by moving 3 glucose from branch to end.
Alpha-glucosidase removes the final glucose from the branch.
Phosphorylase continues.
Alpha-1,6-glucosidase rxn
Phosphorylase rxn

29. Glycogen Synthesis
Glycogen starts from a glycogenin dimer, which synthesizes an a-1,4-linked glucose chain linked to a tyrosine.
Glycogen synthase adds more Glc to this chain from UDP-Glc.
A branching enzyme transfers 7 Glc residues from a chain at least 11 Glc long to make each branch.
Glycogen synthase

30. Glycogen metabolism regulation
Either epinephrine (in muscles) or glucagon (in liver) can stimulate adenylate cyclase.
Hormone stimulated phosphorylation of phosphorylase activates it, increasing glycogen breakdown.
Hormone stimulated phosphorylation of Glycogen synthase inactivates it, decreasing glycogen synthesis.
Phosphatase I can take these phosphates off, reversing the effects. It is stimulated by insulin.

31. Starch synthesis occurs in plastids
Starch is synthesized from ADP-Glc
In chloroplasts, Glc is made from triose phosphates produced in the Calvin cycle.
3-phosphoglycerate phosphorylated to 1,3-bisphosphoglycerate with ATP
1,3-bisphosphoglycerate reduced to Glyceraldehyde-3-P (GAP) with NADPH.
Triose phosphate isomerase converts half of GAP to Dihydroxyacetone phosphate (DHAP)
Aldolase converts GAP + DHAP to Fructose-1,6-bisphosphate (F1,6BP)
F1,6BP is dephosphorylated to Fructose-6-P > Glc6P> Glc1P.

32. Overall picture of starch synthetic pathway
GAP
DHAP
aldolase
Fructose 1,6-P
Fructose 1,6-bisphosphatase
Fructose 6-P
glucose 6-phosphate isomerase
Glucose 6-P
phosphoglucomutase
Glucose 1-P
ATP
ADP-glucose pyrophosphorylase Pi
3-PG
ADP-Glucose
Pi from exchange of triose phosphates with cytoplasm; 3-PG from photosynthesis.
Glc n
Glcn+1
Slide: Rodjana Opassiri
3-PG = 3-phosphoglycerase, GAP = glyceraldehyde-3-P, DHAP = dihydroxyacetone P

33. Starch synthesis in amyloplast
Amyloplast is white plastid for starch synthesis & storage.
Have ADP-Glc, Glc and Glc-6-P transporters.
Glc-6-P transporter exchanges Glc-6-P for Pi.
ADP-Glc transporter exchanges ADP-Glc for AMP
Hexokinase phosphorylates Glc after transport into the amyloplast.

34. Synthesis of Starch Three enzymes for starch synthesis:
ADP-glucose pyrophosphorylase
Starch synthase
Different isoenzymes elongate amylose chains on amylose and amylopectin.
Soluble isoenzymes add a-1,4-linked glucose to amylopectin
Granule-bound isoenzymes (mainly waxy) add a-1,4-linked glucose to amylose inside starch granules.
Starch-branching enzyme
Moves a-1,4-linked chain to a 6-OH approx. 20 Glc residues from the nonreducing end to make an a-1,6-branch.
Two forms, I & II
Form I adds to amylose & less branched amylopectin
Form II adds more shorter branches to highly branched amylopectin.
Different starches have different distances between branches and proportions of amylose to amylopectin.

35. Synthesis of amylose and amylopectin

37. Summary of Glycogen & Starch Synthesis & Breakdown
Glycogen and starch synthesis both involve synthesis of a-(1,4)-glucosyl chains and transferring to make a-(1,6)-linked branches.
Glycogen synthesis uses UDP-Glc as the precursor, while starch synthesis used ADP-Glc.
Phosphorylase breaks down glycogen to Glc-1-P with help from debranching enzyme and a-glucosidase.
Alpha-glucosidase produces glucose, rather than glucose-1-P
Breakdown of starch can be similar or be done by hydrolytic amylases.
Synthesis & breakdown must be carefully controlled to make sure only one happens at one time.