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Biochemistry
The Calvin Cycle and
Pentose Phosphate Pathway
Stryer Ch. 20
Calvin Cycle (Dark Reaction)
• Reduce carbon dioxide to
carbohydrates using NADPH
and ATP produced during the
light reactions of
photosynthesis
• Three Stages
– Fix (attach) CO2 to Ribulose
1,5-bisphosphate forming two
molecules of 3phosphoglycerate
– Reduce 3-phosphoglycerate to
form hexose carbohydrates
– Regenerate ribulose 1,5bisphosphate
Ribulose 1,5-bisphosphate carboxylase /
oxygenase (Rubisco)
• Structure
– 8 large (L) subunits – 55 kd
• 1 catalytic site / L subunit
• 1 regulatory site / L subunit
– 8 small (S) subunits – 13 kd
• Most abundant enzyme on earth.
– ~ 30% of leaf protein
• Reaction rate is slow
– Rate = 3 /s
• ΔG °´= -51.9 kJ/mol (-12.4 kcal/mol)
Rubisco (cont’)
• Rubisco activase catalyzes the
formation of the carbamate.
– Carbamate will form
spontaneously but at a slower
rate.
• ε amino group of R group of
lysine 201reacts with carbon
dioxide.
– Forms carbamate
• Carbamate binds Mg2+ as part
of the metal center
Rubisco (cont’)
• Metal center
– Mg2+ is required for
catalysis
• Mg2+ attached to
Rubisco via:
– Glu
– Asp
– Lys carbamate
» Requires CO2
other than
substrate
carbon
dioxides.
– Also binds water
– Ribulose 1,5bisphosphate binds
and activates
Rubisco
(cont’)
• Carboxylase Activity
– Fix CO2 (1C) to Ribulose 1,5bisphosphate (5C) producing a
unstable intermediate (6C)
which reacts to form 2
molecules of 3phosphoglycerate.
– Reactants
– Ribulose 1,5-bisphosphate
• In Stroma (25°C)
• Carbon dioxide
– [CO2]= 10 μM
– Products
– [O2]= 250 μM
• 3-phosphoglycerate (2
– Favored over oxygenase activity
molecules)
Rubisco (cont’)
•
Oxygenase Activity
– In addition to the carboxylase activity
(adding CO2), Rubisco can also function
as an oxygenase (adds O2)
– Reactants
• Ribulose 1,5-bisphosphate
• oxygen
– Products:
•
Phosphoglycolate (2C) metabolic dead
end
• 3-phosphoglycerate (3C)
•
Oxygenase activity ↑ as temperature ↑.
– C4 and CAM plants use different mechanisms
to overcome this “problem” allowing them to
grow efficiently in hot climates.
Photorespiratory Reactions
• Recovers the carbon
skeleton from
phosphoglycolate
• Chloroplast
Phosphotase
• Peroxisome (aka
microsome)
• Mitochondria
Glycolate
oxidase
Reduction of 3phosphoglycerate to a
hexose requires:
• Reactants
– 3-phosphoglycerate (2 molecules)
• Product
– Fructose 6-phosphate (1 molecule)
inter-converts into other hexoses.
• Energy provided by light
reaction
– ATP
– NADPH
Reduction of 3-phosphoglycerate to a
hexose requires:
• Transketolase
– Transfers a COCH2OH (2C)
unit from a ketose to an
aldose.
– TPP as coenzyme
• Aldolase
– (Do you remember
ALDOLASE? An aldol
condensation)
– Transfers DHAP to an aldose of
(n carbons) making a ketose (n
+3C)
• Specific for DHAP but accepts
many aldehyde containing
sugars
Synthesis of C4, C5 and C7 sugars
• The
combined
action of
transketolase
and aldolase
as part of the
Calvin cycle
provide the
plant with a
source of C4,
C5 and C7
sugars.
Regenerate of ribulose 1,5 bisphosphate
• Phosphopentose
isomerase
• Phosphopentose
epimerase
• Phosphoribulose
kinase
•
1 molecule glucose
requires
– 12 ATP
– 12 NADPH
•
Regeneration of R 1,5
P
– 6 ATP
Synthesis of Starch
• Polymer of glucose with alpha 1-4
glycosidic bonds and alpha 1-6
branches.
– Fewer branches than glycogen
– Activated precursor ADP-glucose
Synthesis of Sucrose
• synthesised in
cytoplasm
• Sucrose-6-phosphate
synthase
– fructose -6 phosphate
• Synthesized from
trioses (G3P) from ct
– Phosphate
translocator
» Antiports
phosphate / G3P
– UDP-glucose
Regulation of the Calvin Cycle
• Light Reaction alters chemistry
of stroma by:
– ↑ [NADPH]
• Reduction of NADP+ by FdNADP+ reductase
• Activates two enzymes by
displacing the inhibitory protein
CP12
– Phosphoribulose kinase
– Glyceraldehyde 3-phophate
dehydrogenase
– ↑ pH from 7 to 8
• Pumping H+ by cyt bf
• Alkaline pH favors carbamate
formation
– ↑ [Mg2+]
• Transported due to transport of H+
– ↑ reduced Fd
• Reduces thioredoxin
Thioredoxin: Structure
• 12 kd protein
• Pair of cysteine cycle between oxidized
disulfides and reduced sulfhydryls
• Contains 4Fe-4S cluster which couples
two single e- reduction into a two ereduction.
Regulation by Thioredoxin
• Activates biosynthetic
pathways by reducing
regulatory enzymes of that
pathway.
• Ferridoxin reduces
thioredoxin which reduces
regulatory enzymes in
pathway.
The Location or Timing of the Calvin
cycle relative to Carbon Fixation reduces
Rubiso’s Oxagenase activity
• (WHO)
– C3, C4, CAM plants
• (WHAT)
– first molecule formed from
CO2
• (WHEN)
– molecule formed
• (WHERE)
– molecule formed
Location/ Timing of Calvin cycle
Differs in C3, C4, and CAM Plants
• (WHY)
– to increase the local
[CO2] relative to [O2]
• favors photosynthesis
over photorespiration.
• Atmosphere is:
– 21% oxygen
– 0.036% carbon dioxide
C3 Plant
(This is what we have been discussing)
• Wheat, rice, oats, rose, soybeans
• 3PG = 3 phosphoglycerate first molecule formed
in Calvin Cycle
RuBP Carboxylase
RuBP + CO2
• Mesophyll cells

– Carbon Dioxide fixation
– Calvin cycle
3 PG
C4 Plants
• Corn, Sugar cane, Bermuda grass.
• Oxaloacetate (C4)
Phosphoenolpyruvate Carboxylase
PEP (C3) + CO2  Oxaloacetate (C4)
Pineapple
CAM
• Spatal (space/ location) separation
CO2
Night
Organic acid
– mesophyll cells
• carbon dioxide fixation
– bundle sheath cells
• Calvin cycle
CO2
Day
Calvin
Cycle
Sugar
(b) Temporal separation of steps
CAM
(Crassulacean Acid Metabolism)
• Succulent desert plants (cacti), pineapple
• Oxaloacetate(C4)

Malate
(C4)
Sugarcane
C
CO
• Temporal (time) separation
4
2
Mesophyll
cell
Organic acid
– Mesophyll cells
1
• night
–Bundlestomata openCO-2 allows CO2 to enter
sheath
cell
2
– carbon fixation
Calvin
• day
Cycle
– stomata closed
Sugar- conserves water
– Calvin
cycle
(a) Spatial
separation of steps
Pentose Phosphate Pathway
• AKA
– Hexose Monophosphate Pathway
– Phosphoglyconate Pathway
– Pentose Monophosphate Shunt
• Function:
– Synthesis of NADPH
• Reduction power for biosynthetic reactions.
– Catabolism / synthesis of C 5 (pentoses)
carbohydrates
• Nucleotide biosynthesis
– Catabolism / synthesis of C 4 (tetroses) and 7 C
(heptoses) carbohydrates
– Linked to glycolysis
• Divided into
– Oxidative phase
• Synthesis of NADPH
– Nonoxidative phase
• Interconversion of C4, C5, C6, and C7
carbohydrates
Pentose Phosphate Pathway
• Location of Pentose phosphate pathway to provide
NADPH:
– Synthesis
•
•
•
•
•
•
•
•
Adrenal gland - Steroid synthesis
Testes - Steroid synthesis
Ovary - Steroid synthesis
Mammary gland - Fatty acid synthesis
Liver - Fatty acid biosynthesis and Cholesterol biosynthesis
Adipose tissue - Fatty acid synthesis
Various tissue - Neurotransmitter biosynthesis
Almost all tissue - Nucleotide biosynthesis
– Detoxification
• Reduction of oxidized glutathione
• Cytochrome P 450 monooxygenase
Enzymes of Pentose phosphate pathway
Oxidative Phase
Pentose Phosphate Pathway
• Produces 1 Ribulose 5 – phosphate and reduces 2
NADPH / glucose 6-phosphate
• Ribulose 5-phosphate (C5) produces
– Ribose 5-phosphate (C5)
– Xylulose 5-phosphate (C5)
Glucose 6-phosphate dehydrogenase
•
•
•
•
Produces 1 NADPH / Glucose 6- phosphate oxidized
Irreversible
Dehydrogenation
Inhibited by ↓ [NADP+] (required to accept electrons)
R
Lactonase
• Hydrolysis
6-Phosphogluconate dehydrogenase
• Produces 1 NADPH / Glucose 6- phosphate oxidized
Phosphopentose isomerase
• Ribulose 5-phosphate → Ribose 5-phosphate
Phosphopentose epimerase
• Ribulose 5-phosphate → xylulose 5-phosphate
Transketolase
• Transfers a COCH2OH (2C) unit from a ketose to an aldose.
• TPP as coenzyme
• C5 + C5 ⇌ C3 + C7
Transketolase (cont’)
• Mechanism similar to E1 subunit of
pyruvate dehydrogenase complex.
Transaldolase
• Transfers 3C units
• Uses R group of lysine to form Schiffs base (no prosthetic group)
• C3 + C7 ⇌ C6 + C4
Transaldolase (cont’)
• Mechanism similar to fructose 1, 6
bisphosphate aldolase in glycolysis.
Transketolase
• C4 + C5 ⇌ C6 + C3
Similarities in Transketolase and
Transaldolase Mechanism
• Both enzymes
produce carbanions
stabilized by
resonance during
catalysis.
– Transketolase
• TPP
– Transaldolase
• lysine
Pentose Phosphate Pathway adjusts to
needs of the cell
• For
production of
NADPH or
various
carbohydrates
Situation 1
High demand for ribose 5-phosphate (for DNA synthesis);
low demand for NADPH
• Glycolysis
linked to
transketolase
and
transaldolase
– 2 molecules
of fructose 6phosphate + 1
molecule of
glyceraldehy
de 3phosphate
→3
molecules
ribose 5phosphate.
Situation 2
Balanced need for ribose 5-phosphate and NADPH.
• Uses oxidative
phase of
pentose
phosphate
pathway to
produce 2
NADPH and 1
ribose 5phosphate.
Situation 3
More NADPH than ribose 5 phosphate required.
• Glucose 6phosphate
completely
oxidized to CO2
• Three reactions
– Oxidative phase
of pentose
phosphate
pathway
– Tranketolase and
tranaldolase
– Gluconeogenesis
Situation 4
Both NADPH and ATP required
• Oxidative phase of
pentose phosphate
pathway.
• Ribose 5-phosphate
converted into F6P
and G3P which enter
glycolysis
• Pyruvate
– Oxidized
– Used as precursors
Glutathione protects against
Reactive oxygen species (ROS)
• Glutathione
– Tripeptide of ECG
• Free sulfhydryl
• GSH → GSSG
– GSH = reduced glutathione
– GSSG = oxidized glutathione
• Glutathione reductase
– Uses NADPH to reduce GSSG →
GSH.
– Contains FAD
• In vivo
– [glutathione] = ~ 5mM
– [GSH] : [GSSG] 500 : 1
Glucose 6phosphate
dehydrogenase
deficiency
• Glucose 6-phosphate dehydrogenase
• X linked inheritance
• MOST common genetic defect
• Pamaquine
– Purine glycoside isolated from a plant
(fava bean)
– Antimalarial drug
– In vivo pamaquine generates
↑
[peroxides]
– causes severe symptoms in some
patients
• Black urine
• Jaundice
• Hemolytic anemia
• Symptoms due to glucose 6phosphate deficiency
– Does not produce NADPH via pentose
phosphate pathway
– NADPH needed by glutathione
reductase to reduce glutathione
• Glutathione destroys ROS in RBCs
• RBCs with Heinz bodies.
– Defining characteristic of glucose 6phosphate dehydrogenase deficiency
– Denatured proteins adhering to plasma
membrane of RBC.