Latiyah Timothy

816012983
Bioc 2061: Bioenergetics
Tutorial 9: Photosynthesis #2

1. RuBisCo activation Mechanism:

 The enzyme consists of 8 large subunits and 8 small subunits. The large subunits contain the
active site of the enzyme.
 Enzyme is inactive because ribulose 1, 5-bisphosphate is bound to the active site. This results
in the closed conformation which makes the lysine side chain for carbamylation inaccessible.
 Activation is catalyzed by rubisco activase by promoting the ATP-dependent release of the
ribulose 1,5-bisphosphate and the lysine side chain is exposed for carbamylation.
 Activation requires light, CO2, Mg2+ and correct stromal pH.
 A non-substrate CO2 adds to the ε-amino group of lysine 201 to form a carbamate.
 Mg2+ binds to the negatively charged carbamate and is linked to rubisco via an oxygen in the
carbamate on Lys201 and two in the carboxyl groups of Glu204 and Asp203
 The enzyme is now active.

2. Formation of 3-Phosphoglycerate by RuBisCo carboxylase:

 Ribulose 1, 5-bisphosphate binds to Mg2+ through its keto group and an adjacent hydroxyl
group.
 This complex is readily deprotonated to form an enediolate intermediate.
 The intermediate reacts with a CO2 to form 2-carboxy-3-keto-D-arabinitol 1,5- bisphosphate.
 A molecule of H2O is then added to this β- ketoacid to form an intermediate that cleaves to
form two molecules of 3-phosphoglycerate.
3. The Reduction and the Regeneration stages in the Calvin (Benson -Basshum) Cycle.

 Reduction: Conversion of 3-Phosphoglycerate to Glyceraldehyde 3-Phosphate

 Stromal 3-phosphoglycerate kinase catalyzes the transfer of a phosphoryl group from
ATP to 3-phosphoglycerate, to produce 1,3-bisphosphoglycerate.
 NADPH donates electrons in a reduction catalyzed glyceraldehyde 3-phosphate
dehydrogenase, producing glyceraldehyde 3-phosphate and Pi.
 Regeneration: Regeneration of Ribulose 1,5-Bisphosphate from Triose Phosphates.
 Interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate by
triose phosphate isomerase.
 Condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate
catalyze by the enzyme aldolase to produce fructose 1,6-bisphosphate.
 Fructose 1,6-bisphosphate is cleaved to fructose 6-phosphate and Pi by fructose 1,6-
bisphosphatase.
 Glyceraldehyde 3-phosphate accepts a keto group from fructose 6-phosphate in which
the transfer is catalysed by transketolase to yield xylulose 5-phosphate and tetrose
erythrose 4-phosphate.
 Erythrose 4-phosphate is combined with dihydroxyacetone phosphate by aldolase to
form sedoheptulose 1,7-bisphosphate.
 Sedoheptulose 1,7-bisphosphate is converted to sedoheptulose 7-phosphate by
sedoheptulose 1,7-bisphosphatase.
 Transketolase converts sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate to
ribose 5-phosphate and xylulose 5-phosphate.
 Ribose 5-phosphate is converted to Ribulose 5-phosphate by phosphopentose
isomerase. While xylulose 5-phosphate is converted to ribulose 5-phosphate by
phosphopentose epimerase.
 Ribulose 1,5-bisphosphate is then regenerated from ribulose 5-phosphate by
phosphoribulose kinase.
4. The Calvin Cycle is regulated at the steps that consume ATP and NADPH, and the site of CO 2
fixation. The enzymes that a regulated are Fructose 1,6-bisphosphatase, Sedoheptulose 1,7-
bisphosphatase, Phosphoglycerate kinase, Phosphoribulokinase, Glyceraldehyde 3-phosphate
dehydrogenase and Rubisco.

 Rubisco, fructose 1,6-bisphosphatase and sedoheptulose 1,7,-bisphosphatase are regulated by

the stromal concentrations of Mg2+ and pH. These enzymes require a high pH and a high
concentration of Mg2+ to activate. These conditions are a result of the light-driven pumping of
protons into the thylakoid space.
 Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase are stimulated by
NADPH. In the dark, these enzymes are inhibited by association with CP12. NADPH disrupts
this association by promoting the formation of two disulfide bonds in CP12, leading to the
release of the active enzymes.
 Thioredoxin plays a role in regulation. Reduced thioredoxin activates certain Calvin cycle
enzymes by cleaving regulatory disulfide bonds.
5. RuBisCo has an oxygenase activity.

 It is no specific for CO2 only. During evolution, O2 levels were lower and there was no
selective pressure to discriminate between CO 2 and O2.
 Oxygen competes with CO2 at the active site.
 About once in every three or four turnovers, rubisco catalyzes the condensation of O 2 with
ribulose 1,5-bisphosphate.
 This reaction forms 3-phosphoglycerate and 2-phosphoglycolate. The 2-phosphoglycolate is
metabolically useless.
 Hence the full name of the enzyme is ribulose 1,5-bisphosphate carboxylase/oxygenase.
 This oxygenase is energetically demanding on the cell as carbons have to be salvaged from 2-
phosphoglycolate via photorespiration.

6. C3 and C4 plants overcome the oxygenase activity by photorespiration and increasing the
concentration of CO2 in photosynthetic cells, respectively.

 C3 plants and Photorespiration: Glycolate pathway

 2-phosphoglycolate phosphatase converts 2-phosphoglycolate to glycerate
(chloroplast)
 Glycolate enters the peroxisome and is oxidised to glyoxylate by glycolate oxidase.
 Glyoxylate undergoes transamination to glycine. The hydrogen peroxide formed as a
side product of glycolate oxidation is rendered harmless by peroxidases in the
peroxisome.
 Glycine passes from the peroxisome to the mitochondrial matrix, where it undergoes
oxidative decarboxylation by the glycine decarboxylase complex. This complex is
similar to the PDH complex and α-ketoglutarate dehydrogenase complex.
 The glycine decarboxylase complex oxidizes glycine to CO2 and NH3, with the
concomitant reduction of NAD+ to NADH and transfer of the remaining carbon from
glycine to the cofactor tetrahydrofolate. The one-carbon unit carried on
tetrahydrofolate is then transferred to a second glycine by serine
hydroxymethyltransferase, producing serine.
  The serine is converted to hydroxypyruvate, then to glycerate, and finally to 3-
phosphoglycerate, which is used to regenerate ribulose 1,5-bisphosphate.
 This salvage pathway serves to recycle three of the four carbon atoms of two
molecules of glycolate

 C4 plants: C4 pathway
 CO2 Fixation and Rubisco Activity Are Spatially Separated.
 Involves mesophyll and bundle sheath cells.
 In the mesophyll cell, CO2 is condensed with phosphoenolpyruvate to form
oxaloacetate in a reaction catalyzed by phosphoenolpyruvate carboxylase.
 Phosphoenolpyruvate carboxylase has a lower Km for CO 2 and, hence, higher
affinity than Rubisco.
 O2 is a poor substrate for this enzyme, there even at low concentrations of CO 2, CO2 is
still fixed.
 In some species, oxaloacetate is converted into malate or aspartate by an NADP +-
malic enzyme(
 Malate/aspartate enters the bundle-sheath cell and is oxidatively-decarboxylated
within the chloroplasts by an NADP+-malic enzyme
 The released CO2 enters the Calvin cycle in the usual way by condensing with
ribulose 1,5-bisphosphate.
 Pyruvate formed in this decarboxylation reaction returns to the mesophyll cell.
 phosphoenolpyruvate is formed from pyruvate by pyruvate-Pi dikinase.
7. C3 plants reassimilate the nitrogen lost at glycine decarboxylase by Nitrogen Fixation via the
GS/GOGAT pathway.

 The glutamine synthetase (GS) fixes ammonium on a glutamate molecule to form glutamine.
 This glutamine reacts subsequently with 2-oxoglutarate to form two molecules of glutamate,
this step being catalysed by the glutamine 2-oxoglutarate amino transferase (or glutamate
synthase, GOGAT)
 α-Ketoglutarate, undergoes reductive amination with glutamine to produce glutamate

8. In CAM plants, CO2 Capture and Rubisco Action Are Temporally Separated

 Occurs in succulents, that are in arid conditions.

 Trapping of CO2 and its fixation by RuBisCo is separated by time of day.
 At night when the air is cooler and moister, the stomata open to allow entry of CO 2, which is
then fixed into oxaloacetate by PEP carboxylase.
 The oxaloacetate is reduced to malate and stored in the vacuoles.
 During the day the stomata close, preventing the water loss that would result from high
daytime temperatures, and the CO2 trapped overnight in malate is released as CO 2 by the
NADP-malic enzyme.
 This CO2 is now assimilated by the action of rubisco and the Calvin cycle enzymes.