Lecture 13 Summary

The Calvin cycle assimilates CO₂ into carbohydrates. When CO₂ is fixed in the reaction catalyzed by RuBisCO, the 3-phosphoglycerate that is formed is converted to glyceraldehyde 3-phosphate (GAP). This production phase uses ATP and NADPH and yields two glyceraldehyde 3-phosphate (GAP) for each CO₂ and ribulose 1,5-bisphosphate combined in the RuBisCO carboxylation reaction. Since GAP is an intermediate of both glycolysis and gluconeogenesis, is can be used to produce fructose 6-phosphate. A regeneration phase recombines 3- and 6-carbon sugars to form ribulose 5-phosphate (Ru5P), which is phosphorylated using ATP to finally regenerate ribulose 1,5-bisphosphate (RuBP). Out of every 6 GAP molecules produced, 5 are recycled in the regeneration of RuBP, and one formally represents the net production of a 3-carbon sugar from three CO₂ molecules.

Enzymes participating in the Calvin cycle

Enzyme [abbreviation, EC number] - pathway (other than Calvin cycle)

• Ribulose 1,5-bisphosphate carboxylase [RuBisCO, EC 4.1.1.39] - (specific to Calvin cycle)
• Phosphoglycerate kinase [PGK, EC 2.7.2.3] - gluconeogenesis/glycolysis
• Glyceraldehyde 3-phosphate dehydrogenase [GAPDH, EC 1.2.1.12] - gluconeogenesis/glycolysis
• Triose phosphate isomerase [TIM, EC 5.3.1.1] - gluconeogenesis/glycolysis
• Transaldolase [TA, EC 2.2.1.2] - pentose phosphate pathway; aldolase [EC 4.1.2.13] - gluconeogenesis/glycolysis
• Fructose 1,6-bisphosphatase [FBPase, EC 3.1.3.11] - gluconeogenesis
• Transketolase [TK, EC 2.2.1.1] - pentose phosphate pathway
• Sedoheptulose 7-phosphatase [SBPase, EC 3.1.3.37] - (specific to Calvin cycle)
• Phosphopentose isomerase [EC 5.3.1.6] - pentose phosphate pathway
• Phosphoribulose epimerase [EC 5.1.3.1] - pentose phosphate pathway

Transketolase

• Common to both Calvin cycle and pentose phosphate pathway
• TPP cofactor
• Transfer of a 2-carbon fragment from an aldose to a ketose via an activated glycoaldehyde intermediate :
  • ketose_m + aldose_n = aldose_m–2 + ketose_n+2

Aldolase and Transaldolase

• Transaldolase is common to both Calvin cycle and pentose phosphate pathway
• Class I aldolase: active site Lys forms Schiff base linkage with substrate.
• Transaldolase: Transfer of a 3-carbon fragment from a ketose to an aldose to form new aldose/ketose pair :
  • aldose_m + ketose_n =  ketose_m–2 + aldose_n+2
• In aldolase, the ketose substrate is DHAP, and there is no aldose product

Transketolase and transaldolase mechaisms

Resonance-stabilized intermediates that react like carbanions toward electrophilic carbonyl carbons are formed in both the transketolase [EC 2.2.1.1] and the transaldolase [EC 2.2.1.2] mechanisms.

Regulation of the Calvin cycle

The output of the light reactions of photosynthesis - ATP, NADPH - are utilized by the Calvin cycle. The rate at which the Calvin cycle operates is coupled to the operation of the light reactions in several ways. The pH, magnesium concentration, and redox status of the stroma all play a role.

The pH of the stroma increases upon illumination of the chloroplast, due of course to the photo-driven H⁺-transport across the thylakoid membrane. Furthermore, illumination drives electrons toward ferredoxin (Fd) and NADP⁺ so that their reduced forms predominate. Third, illumination triggers a release of Mg²⁺ from the thylakoid lumen to the stroma. These three factors contribute to an increase in the activity of Calvin cycle enzymes. Hence, the flux of metabolites through the Calvin cycle is increased, and more carbohydrate is produced.

Since enzymes typically display pH optima, it is to be expected that the enzymes of the Calvin cycle would have optimum activity at a pH around 8. As the pH increase is driven by illumination, this is a simple means of coupling, or coordinately regulating, the light and dark reactions of photosynthesis. We have already explained the dependence of RuBisCO activity on magnesium. The redox status of the stromal environment is indicated by the ratios [NADPH]/[NADP⁺] or [Fd]_red/[Fd_ox]. If these ratios are high, then the stroma is at a releatively high reduction potential. This is somewhat akin to having a charged battery. A protein called thioredoxin couples the activities of Calvin cycle enzymes to the stromal reduction potential, by a "covalent" regulatory mechanism.

A ubiquitous disulfide redox protein, thioredoxin (molecular weight 12 kDa) contains a pair of cysteine residues that alternate between oxidized and reduced forms. In the oxidized form, the two cysteines are crosslinked, joined by an internal disulfide bridge. In the reduced form of thioredoxin, the disulfide bond is cleaved, leaving both cysteines in their sulfhydryl (-SH) forms. Like ferredoxin, the nicotinamide cofactors, and cytochrome c, thioredoxin cycles between redox states. This cycling mediates a redox switch that regulates activites of Calvin cycle enzymes. Note in the ribbon diagram shown at right the disulfide link between Cys46 and Cys49 (yellow) in the upper left, near a surface loop (cyan) at the start of an a-helical stretch (red). Thioredoxin can undergo disulfide exchange with other proteins with similar pairs of cysteine residues. In particular, the Calvin cycle enzymes RuBisCO [EC 4.1.1.39].and sedoheptulose bisphosphatase [EC 3.1.3.37] are regulable by their own redox switches. The reduced form of thioredoxin activates those enzymes by undergoing disulfide exchange with a target enzyme.

The biochemical standard free energy change for the carboxylation reaction catalyzed by RuBisCO is –12.4 kcal mol^-1. This is a significant drop in free energy, and since RuBisCO carboxylase reaction represents the first step of photosynthetic carbon assimilation, it is the committed step. Enzymes catalyzing the committed step of a pathway are generally highly regulated, and RuBisCO is no exception.

Since RuBisCO requires Mg²⁺ for activity, a simple way to regulate the enzyme would be to control the availability of Mg²⁺ and affinity of the enzyme for the cation. In chloroplasts exposed to light and carrying out the light reactions of photosynthesis, the stromal pH increases, and Mg²⁺ is transported from the thylakoid lumen to the stroma. Upon illumination, the stromal pH typically increases from ~ 7 to ~ 8, and the Mg²⁺ concentration increases from 1 - 3 mM to 3 - 6 mM. An interesting regulatory mechanism exists for RuBisCO that effectively couples its activation to these light-induced changes as well as to the availability of its substrate CO₂. In the dark, RuBisCO actually exists in an inactive, "closed" conformation. In this closed conformation, the substrate RuBP is tightly bound, but Lys201 remains unmodified since it is inaccessible in this conformation, and correspondingly, Mg²⁺ is not bound since the carbamoylated Lys201 is a critical ligand for Mg²⁺. An ATP-dependent enzyme known as rubisco activase uses the energy of ATP to promote the release of RuBP and favoring a conformational change in RuBisCO exposing Lys201. Then in the presence of CO₂ and high pH, Lys201 becomes carbamoylated, facilitating Mg²⁺-binding. Interestingly, rubisco activase is itself regulated in some species by a thioredoxin-dependent redox mechanism.

Calvin cycle adaptations: The C4 pathway and CAM.

C4 pathway

The oxygenase activity of RuBisCO increases more rapidly with temperature than does the carboxylase activity. The C4 pathway is an adaptation common in plants living in hot climates that uses energy to fix CO₂ in the form of malate in mesophyll cells. Malate is then transported to the chloroplast stroma of bundle sheath cells, where it is oxidatively decarboxylated by NADP⁺-malic enzyme [EC 1.1.1.40]. This generates carbon dioxide in situ at a relatively high concentration, thereby increasing the efficiency of RuBisCO as the oxygenase reaction is competitively suppressed.

CAM plants

The so-called CAM plants - primarily succulents living in very arid climates (CAM stands for Crassulacean acid metabolism) - have adapted to the inefficiency of RuBisCO in a different way, by temporal segregation of CO₂ uptake and fixation as malate and its combination with ribulose 1,5-bisphosphate via the carboxylase reaction of RuBisCO.

Study questions

• Explain why the phrase "dark reactions" is a potentially misleading one.
• Work out the path that regenerates ribulose 5-phosphate from 3-phosphoglycerate. Use the activities of TA, TK, SBPase, and any enzymes of glycolysis or gluconeogenesis as required.
• Name four factors controlling the rate of the Calvin cycle and describe their mechanism, logic, and significance.
• Explain the mechanisms of regulation of RuBisCO by pH, Mg²⁺, and thioredoxin.
• Explain the tradeoffs for C4 pathway or CAM in comparison with the nomal Calvin cycle.

Page updated 12-27-06

References

1. Berg, Tymoczko, and Stryer. Biochemistry (BTS): 6th edition (2007, Freeman) Ch.20, pp.570-577.