Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3020
III. Metabolism
Carbohydrate Synthesis
CO2 Assimilation
Autotrophic plants convert CO2 into organic compound (triose phosphates)
Biosynthesis occures in chlorpolsts
CO2 fixation requires ATP and NADPH
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Carbon Assimilation
The Calvin cycle
→ cyclic three step
process
CO2 is condensed with
5C-sugar (R15BP) to
yield two 3C sugars
G3P is formed from
R15BP on the expense
of ATP and NADPH
Rearrangement of six
3C sugars to three 5C
sugars.
→ regeneration of R15BP
Carbon Fixation - RUBISCO
Carbon fixation requires the enzyme
Ribulose 1,5-bisphosphate carboxylase/
oxigenase → RUBISCO
Covalently attaches CO2 to R15BP
Cleaves 6C to two molecules
of 3-phosphoglycerate
Spinach RUBISCO
Has an undesirable oxygenase
activity.
Most abundant protein in the known universe.
Bacterial RUBISCO is simpler.
→ subunit structure that of the plant enzyme
Bacterial RUBISCO
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Catalytic Mechanism of RUBISCO
RUBISCO needs to be activated by
the addition of CO2 to an active-site
Lysine.
ATP hydrolysis is used to displace
R15BP. CO2 is added to Lys 201
→ Mg2+ can bind.
PDBid 1RXO
Activation is catalyzed by
RUBISCO activase
Catalytic Mechanism of RUBISCO
The carboamylated lysine, stabilized by Mg2+, abstracts
a H+ from the central carbon of R15BP.
→ generation of an endiole intermediate
→ attacks the CO2
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Catalytic Mechanism of RUBISCO
Mg2+ plays a central role in
the catalytic mechanism at
all stages.
RUBISCO inhibitor
Why?
Transition state
analog
Calvin Cycle – Stage 2
The second stage is a reduction
→ set of two reaction
Reversal of the equivalent steps
in glycolysis
→ BUT NADPH is the cofactor
Chloroplast stroma contains all
glycolytic enzymes except
phosphoglycerat mutase
Stromal and cytosolic enzymes are isozymes, they catalyze the same reaction
but they are products of different genes.
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Calvin Cycle – Stage 2
The Fate of Glyceraldehyde 3-phosphate
Triose phosphate isomerase
converts some Glyceraldehyde
3-phosphate to dihydroxyaceton
phosphate.
→ can enter gluconeogenesis
or glycolysis
The majority of glyceraldehyde 3-phosphate is converted to R15BP
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Calvin Cycle – Stage 3
The third stage is the
regeneration of the acceptor
→ several reaction
Calvin Cycle – Stage 3
Regeneration of Ribulose 1,5-Bisphosphate from triose phosphates.
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Calvin Cycle – Stage 3
Regeneration of Ribulose 1,5-Bisphosphate from triose phosphates.
Do you remember that one?
Pentose Phosphate Pathway
Alternative path to oxidize
Glucose.
The electron acceptor is NADP+.
NADPH is needed for reductive
biosynthesis.
Products are pentose phosphates.
High amounts of NADPH prevent
oxidative damage to proteins
(red blood cells, cornea).
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Nonoxidative Reactions of the Pentose
Phosphate Pathway
5 hexoses (6C) are
made from 6 pentoses (5C)
Every reaction shown here is reversibel !
Transketolase
Transketolase
→ transfer of 2-carbon group
→ TPP-mediated
Reaction important for the Calvin-Cycle:
1. Conversion of hexoses
and trioses to a four- and
a five-carbon sugar
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Transketolase
Reaction important for the Calvin-Cycle:
2. Conversion of seven-carbon and three-carbon sugars to two pentoses.
Regeneration of ribulose 1,5-bisphosphate
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Calvin Cycle Stoichiometry
6 NADPH and 9 ATP are
required to produce
1 glyceraldehyde 3phosphate → 2:3 ratio
Results in the net loss of
1 Pi from the chloroplast
Phosphate Import
Inner chloroplast membrane
is impermeable to most
phosphorylated compounds
What to do?
Inner chloroplast membrane has an antiport system for Pi and triose phosphate.
Glyceraldehyd 3-phosphate is converted to dihydroxyaceton phosphate by
triose phosphate isomerase. → DHAP is exchanged 1:1 for Pi
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What About ATP and NADPH?
ATP and NADPH can not cross the chloroplast membrane.
But the Pi-triose phosphate antiport
system has the indirect effect of
moving ATP and reducing
equivalents across the membrane.
Dihydroxyaceton is
transported to the cytosol
where it is converted to
3-phosphoglycerate (ATP/NADH)
→ glycolytic enzymes
Light Regulation of The Dark Reactions
Any Idea ?
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Light Regulation of The Dark Reactions
Illumination of chloroplasts leads to
→ transport of H+ into the thylakoid
→ results in Mg2+ transport to the stroma
→ increase in NADPH
These signals are used to regulate
stromal enzymes.
Activation of RUBISCO by formation of
the carbamoyllysine is faster at
alkaline pH.
High Mg2+ concentration favor formation
the enzymes Mg2+ active complex.
Light Regulation of The Dark Reactions
Fructose 1,6-bisphosphatase requires Mg2+ and is very dependent on pH.
→ its activity increases more the 100 fold when pH and [Mg2+] rise
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Light Regulation of The Dark Reactions
Light reactions also result in electron flow through ferredoxin. Electrons are
passed to thioredoxin by the enzyme ferredoxin-thioredoxin reductase.
→ thioredoxin activates the carbon assimilation reactions.
Calvin cycle enzymes up-regulated by reduction of disulfides:
→ seduheptulose 1,7 bisphosphatase
→ fructose 1,6 bisphosphatase
→ ribulose 5-phosphate kinase
→ glyceraldehyde 3-phosphate dehydrogenase
Light Regulation of The Dark Reactions
Light reactions also result in electron flow through ferredoxin. Electrons are
passed to thioredoxin by the enzyme ferredoxin-thioredoxin reductase.
→ thioredoxin activates the carbon assimilation reactions.
X
Light reaction stops the enzymes
disulfides are reoxidized.
Calvin cycle enzymes up-regulated by reduction of disulfides:
→ seduheptulose 1,7 bisphosphate
→ fructose 1,6 bisphosphatase
→ ribulose 5-phosphate kinase
→ glyceraldehyde 3-phosphate dehydrogenase
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Photorespiration
RUBISCO is not absolutely specific for CO2 as a substrate.
→ molecular O2 competes with CO2
→about 30% of the reactions consume O2
Why is that a problem ?
Loss of 2C’s from the
Calvin-cycle
Has to be recycled
The Salvage of Phosphoglycolate
The Glycolate Pathway
In cloroplasts a phosphatase
converts 2-phosphoglycolate
to glycolate.
→ exported to peroxisome
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The Salvage of Phosphoglycolate
The Glycolate Pathway
Glycolate is oxidized to
glyoxylate.
Glyoxylate undergoes
transamination to glycine
→ exported to mitochondria
The formed peroxide is deactivated by
peroxidases in the peroxysome.
The Salvage of Phosphoglycolate
The Glycolate Pathway
Two glycine molecules
form Serine and CO2
→ Glycine decarboxylase
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Glycine Deacrboxylase
Have you seen something
similar before?
→ where?
Pyruvate Dehydrogenase Complex
Oxidative decarboxylierung of pyruvate to acetyl-CoA.
Step 1 is rate limiting and responsible for substrate specificity.
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Glycine Decarboxylase
Similar in structure and function
to two mitochondrial complexes
→ pyruvate dehydrogenase
→ α-ketoglutarate dehydrogenase
Glycine decarboxylase:
4 subunits P,H,T & L
→ stoichiometry P4H27T9L2
Step 1
Schiff base formation between
glycine and pyridoxal phosphate (PLP)
Step 2
Protein P catalyzes oxidative decarboxylation
of glycine
Glycine Decarboxylase
Step 3
Protein T releases NH3
from the methylamine
moiety
→ cofactor: tetrahydrofolat (H4T)
Step 4
Protein L oxidized the
two SH-groups of lipoic acid.
Step 5
FAD is regenerated by transfering
electrons ton NADH.
N5,N10-methylen H4F is used by serine
hydroxymethyltransferase to convert one
molecule of glycine to serin.
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CO2 Fixation in C4 Plants
C4 component
Avoiding the wasteful photorespiration some
plants have evolved a separate “C4 pathway”
→ specialized cells
In mesophyll cells CO2 is fixed as HCO3- by
PEP carboxylase.
CO2 is split off by malic enzyme in the bundlesheath cell → high local concentration
→ less O2 missincorporation
CO2 Fixation in C4 Plants
C4 pathway has a greater energy cost
than the C3 pathway.
→ 5 ATP per CO2 vs. 3 ATP in C3
But at higher temperatures the affinity
of RUBISCO for CO2 decreases.
→ at about 28 to 30°C the gain in efficiency
is overcompensated by the
elimination of photorespiration.
→C4 plants outgrow most C3 plants
during summer.
Some plants, native to very hot &
dry environments separate CO2
trapping and fixation over time.
CO2 is trapped and stored as malate
in the night. During day stomata close
and CO2 is released by malic enzyme
→ assimilation via RUBISCO
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Reading
Chapter 42
April 26, 2005
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