Molecular Biochemistry II

Calvin Cycle - Photosynthetic Carbon Reactions

Contents of this page:
Localization of photosynthetic pathways
CO₂ Fixation - role of Ribulose Bisphosphate Carboxylase (RuBisCO)
Photorespiration
Rest of the Calvin Cycle (Carbon Reactions) pathway
Regulation of Calvin Cycle

In the photosynthetic light reactions, energy of light is conserved as as "high energy" phosphoanhydride bonds of ATP, and as reducing power of NADPH.  The proteins and pigments responsible for the photosynthetic light reaction are associated with the thylakoid (grana disk) membranes. The light reaction pathways will not be presented here.

The Calvin Cycle, earlier designated the photosynthetic "dark reactions" pathway, is now referred to as the carbon reactions pathway. In this pathway, the free energy of cleavage of ~P bonds of ATP, and reducing power of NADPH, are used to fix and reduce CO₂ to form carbohydrate. Enzymes and intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat analogous to the mitochondrial matrix. 

Ribulose Bisphosphate Carboxylase (RuBP Carboxylase) catalyzes CO₂ fixation:

ribulose-1,5-bisphosphate + CO₂ à 2 copies of 3-phosphoglycerate

Because it can alternatively catalyze an oxygenase reaction (discussed below), the enzyme is also called RuBP Carboxylase/Oxygenase (RuBisCO). It is the most abundant enzyme on earth. The RuBP Carboxylase reaction mechanism is presented on p. 900.

Extraction of a proton from C3 of ribulose-1,5-bisphosphate (RuBP, below left) promotes formation of an endiolate intermediate. Nucleophilic attack on CO2 is proposed to yield a b-keto acid intermediate, that reacts with water and cleaves to form 2 molecules of  3-phosphoglycerate.

Transition state analogs of the postulated b-keto acid intermediate bind tightly to the enzyme and inhibit its activity. Examples include 2-carboxyarabinitol-1,5-bisphosphate (CABP, at right) and carboxyarabinitol-1-phosphate (CA1P).  RuBP Carboxylase in plants is a complex (L8S8) of:

• 8 large catalytic subunits (L, 477 amino acid residues)
• 8 small subunits (S, 123 amino acid residues). 

Some bacteria contain only the large subunit, with the smallest functional unit being a homodimer, L₂. Roles of the small subunits have not been clearly defined, although there is some evidence that interactions between large and small subunits may regulate catalysis.

At right are 2 views of spinach RuBisCO (RuBP Carboxylase), with large subunits colored blue or cyan, and small subunits colored red.

Large subunits within RuBisCO are arranged as antiparallel dimers, with the N-terminal domain of one monomer adjacent to the C-terminal domain of the other monomer. Each active site is at an interface between  monomers within an L2 dimer, explaining the minimal requirement for a dimeric structure. The substrate binding site is at the mouth of an ab-barrel domain of the large subunit. Most active site residues are polar, including some charged amino acids (e.g., Thr, Asn, Glu, Lys).

"Active" RuBP Carboxylase includes a carbamate group, that binds an essential Mg++ at the active site.  The carbamate forms by reaction of HCO3- with the e-amino group of a lysine residue of RuBP Carboxylase, in the presence of Mg++. HCO3- that reacts to form the carbamate group is distinct from CO2 that binds to RuBP Carboxylase as substrate.  The active site Mg++ bridges between oxygen atoms of the carbamate and the substrate CO2.

Binding of either the normal substrate ribulose-1,5-bisphosphate or a transition state analog to RuBP Carboxylase causes a conformational change to a "closed" conformation in which access of solvent water to the active site is blocked.

RuBP Carboxylase (RuBisCO) can spontaneously deactivate by decarbamylation. In the absence of the carbamate group, RuBisCO tightly binds ribulose bisphosphate (RuBP) or another sugar phosphate at the active site as a "dead end" complex, with the closed conformation, and is inactive in catalysis. In order for the carbamate to reform, the enzyme must undergo transition to the open conformation.

RuBP Carboxylase Activase, an ATP hydrolyzing (ATPase) enzyme, causes a conformational change in RuBP Carboxylase from closed to open form. This allows release of tightly bound RuBP or other sugar phosphate from the active site, and carbamate formation. Since photosynthetic light reactions produce ATP, the ATP dependence of RuBisCO activation provides a mechanism for light-dependent activation of the enzyme.

RuBP Carboxylase Activase is a member of the AAA family of ATPases, many of which have chaperone-like roles. The activase is a large multimeric protein complex that may surround RuBP Carboxylase while inducing the conformational change to the open state.

The complex pathway that partly salvages carbon from 2-phosphoglycolate, via conversion to 3-phosphoglycerate, involves enzymes of chloroplasts, peroxisomes and mitochondria. This pathway recovers 3/4 of the carbon from 2-phosphoglycolate as 3-phosphoglycerate while the rest is released as CO₂. For diagrams see p. 903 and an article by Reumann & Weber.

Photorespiration is a wasteful process, substantially reducing efficiency of CO₂ fixation.

C₃ vs C₄ plants:

Most plants, designated C₃, fix CO₂ initially via RuBP Carboxylase, yielding the 3-carbon compound 3-phosphoglycerate.

Plants designated C4 have one cell type in which phosphoenolpyruvate (PEP) is carboxylated via the enzyme PEP Carboxylase, to yield the 4-carbon compound oxaloacetate. The oxaloacetate is converted to other 4-carbon intermediates that are transported to cells active in photosynthesis, where CO2 is released by decarboxylation. See diagram p. 904.

Continuing with Calvin Cycle: The normal product of the RuBP Carboxylase reaction, 3-phosphoglycerate, is converted to glyceraldehyde-3-phosphate.

• Phosphoglycerate Kinase catalyzes transfer of P_i from ATP to 3-phosphoglycerate, yielding 1,3-bisphosphoglycerate.
• Glyceraldehyde-3-phosphate Dehydrogenase catalyzes reduction of the carboxyl of 1,3-bisphosphoglycerate to an aldehyde, and P_i is released, yielding glyceraldehyde-3-phosphate. This reaction is analogous to Glyceraldehyde-3-phosphate Dehydrogenase of Glycolysis running backward, except that the dehydrogenase within chloroplasts uses NADPH as electron donor, while the cytosolic Glycolysis enzyme uses NAD⁺ as electron acceptor.

A portion of the glyceraldehyde-3-phosphate is converted back to ribulose-1,5-bisphosphate, via reactions catalyzed by Triose Phosphate Isomerase, Aldolase, Fructose Bisphosphatase, Sedoheptulose Bisphosphatase, Transketolase, Epimerase, Ribose Phosphate Isomerase, and Phosphoribulokinase. Many of these enzymes are equivalent to enzymes of the cytosolic Glycolysis, Gluconeogenesis and Pentose Phosphate Pathways, but are separate gene products resident within the chloroplast stroma. (Enzymes of the other pathways listed are located in the cytosol.) The process is similar to the Pentose Phosphate Pathway running backwards.

For three molecules of ribulose-1,5-bisphosphate (total of 15 C) that are carboxylated, cleaved, phosphorylated, reduced, and dephosphorylated, six molecules of glyceraldehyde-3-phosphate are produced (total of 18 C). Of these:

• One glyceraldehyde-3-phosphate (3 C) exits the pathway as product. 
• Five of the 3-C molecules (total of 15 C) are recycled back into three 5-C molecules of ribulose-1,5-bisphosphate, (substrate for RuBisCO), as summarized below.

C3 + C3 à C6 C3 + C6 à C5 + C4 C3 + C4 à C7 C3 + C7 à C5 + C5 Overall: 5C3 à 3C5  Enzymes in the diagram at right: TI = Triosephosphate Isomerase AL = Aldolase FB = Fructose-1,6-bisphosphatase SB = Sedoheptulose-1,7-bisphosphatase TK = Transketolase EP = Epimerase IS = Isomerase PK = Phosphoribulokinase See also diagram p. 897.

Summary of Calvin Cycle, omitting compounds that are regenerated:

3 CO₂ + 9 ATP + 6 NADPH à glyceraldehyde-3-phosphate + 9 ADP + 8 P_i + 6 NADP⁺

Glyceraldehyde-3-phosphate may be converted to other carbohydrates such as metabolites (e.g., fructose-6-phosphate and glucose-1-phosphate), energy stores (e.g., sucrose or starch), or cell wall constituents (e.g., cellulose). Glyceraldehyde-3-phosphate can also be utilized by plant cells as carbon source for synthesis of other compounds such as fatty acids and amino acids. There is evidence for the existence of multienzyme complexes of Calvin Cycle enzymes within the chloroplast stroma. Positioning of many Calvin Cycle enzymes close to the enzymes that produce their substrates or utilize their reaction products may increase efficiency of the pathway.

Light-activated electron transfer is linked to pumping of H⁺ into thylakoid disks. The pH in the stroma increases to about 8. The alkaline pH activates stromal Calvin Cycle enzymes RuBP Carboxylase, Fructose-1,6-Bisphosphatase and Sedoheptulose Bisphosphatase.

The light-activated shift of H⁺ into thylakoid disks is countered by Mg⁺⁺ release from the thylakoids to the stroma. RuBP Carboxylase (in the stroma) requires Mg⁺⁺ binding to carbamate at the active site.

Some plants synthesize the transition-state inhibitor carboxyarabinitol-1-phosphate (CA1P) in the dark. RuBP Carboxylase Activase facilitates release of CA1P from RuBP Carboxylase, when it is activated under conditions of light via thioredoxin.

During illumination, the thioredoxin disulfide is reduced to a dithiol by ferredoxin, a constituent of the photosynthetic light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase. See also diagram p. 902. The reduced thioredoxin activates several Calvin Cycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase Activase, by reducing specific disulfides in these enzymes to thiols.

Copyright © 1998-2008 by Joyce J. Diwan. All rights reserved.

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