Download Carbohydrate Synthesis 1. Photosynthesis

4-1
Lec # 11
Carbohydrate Synthesis
Up to this point in the course, the main focus has been the breakdown of metabolites, including
carbohydrates, lipids and amino acids. The primary purpose of these pathways is to extract
energy in useable form with the common end product being ATP, the "energy currency" of the cell.
In the case of glucose, the break down can be expressed by:
(CH2O)6 + 6 O2
6 CO2 + 6 H2O
∆G'o = -2868 kJ/mol (energy released)
Obviously, the material to be degraded must have originated somewhere and the starting point for
all organic carbon is the fixation of CO2 into carbohydrate via photosynthesis.
1. Photosynthesis - Introduction
To reverse the above reaction such that CO2 is reduced or converted into glucose, energy must be
supplied and in photosynthetic cells light energy is used.
6 CO2 + 6 H2O
(CH2O)6 + 6 O2
∆G'o = +2868 kJ/mol (energy used)
In this one process, not only is reduced carbon (ie. carbohydrate) produced but molecular oxygen,
required for respiration, is produced. When the two processes of photosynthesis and respiration
are combined, the carbon cycle is generated.
carbohydrates
lipids
amino acids
nucleotides
organic carbon
O2
respiration
energy released
(ATP)
photosynthesis
CO2
energy used
(hν or light energy)
4-2
Both procaryotes and eucaryotes are capable of carrying out photosynthesis. While the overall
reactions are similar, there are differences apparent and the following section compares three cases
to highlight the commonalities.
1. Green plants and algae
6 CO2 + 6 H2O
(CH2O)6 + 6 O2
is the usual representation but if 6 H2O are added to both sides we get:
6 CO2 + 12 H2O
(CH2O)6 + 6 O2 + 6 H2O
2. Green sulfur bacteria
6 CO2 + 12 H2S
(CH2O)6 + 12 S + 6 H2O
3. Purple non-sulfur bacteria
6 CO2 + 12 CH3CH(OH)CH3
(isopropanol)
(CH2O)6 + 12 CH3COCH3 + 6 H2O
(acetone)
Comparison of the three overall reactions produces the generalized overall reaction:
6 CO2 + 12 H2X
electron electron
acceptor donor
(CH2O)6 + 12 X + 6 H2O
Reflection on this generalized reaction in relation to the most common reaction from plants and
algae leads to the realization that the oxygen atoms in the product waters must have originated in
the input CO2 while the molecular oxygen (O2) must have originated from the input electron donor
(H2O). In short, there may be two stages to the process as follows:
24 H+ + 24 e- + 12 X
Stage 1
12 H2X
Stage 2
24 H+ + 24 e- + 6 CO2
(CH2O)6 + 6 H2O
Two key experiments addressed and confirmed this idea.
1. The first experiment identified the electron acceptors that became reduced electron carriers
(Hill reagents after the scientist) generated when electrons are removed from H2X (clearly
electrons don't float around loose in solution), and demonstrated that they were generated
independent of CO2.
4-3
Hill found that isolated chlorplasts were capable of generating molecular oxygen (O2) in the
absence of CO2 if they were provided with an electron acceptor. A variety of electron acceptors
were found to work in vitro and these became known as "Hill reagents".
hν
3+
H2O + 2 Fe
1/2 O2 + 2 Fe2+ + 2 H+
donor acceptor
Eventually, it was determined that the actual in vivo electron acceptor in plants was NADP+.
H2O + NADP+
1/2 O2 + NADPH + H+
2. In order to prove that molecular oxygen was derived from the H2O and not from CO2, heavy
water (H218O) was used with the result that only 18O2 was found - no 16O2 (from the C16O2) or
(CH218O)6. Then if the overall reaction is written normally, it is not balanced correctly.
6 C16O2 + 6 H218O
(CH216O)6 + 618O2
However, it can be balanced simply by adding 6 H218O to the left and 6 H216O to the right side.
6 C16O2 + 12 H218O
(CH216O)6 + 618O2 + 6 H216O
This makes it very clear that the input and output waters are treated separately and this is easily
explained by there being two stages to the process as deduced above.
hν
18
+
Stage 1
12 H2 O + 12 NADP
6 18O2 + 12 NADPH + 12 H+
Stage 2
6 C16O2 + 12 NADPH + 12 H+
(CH216O)6 + 6 H216O + 12 NADP+
Because stage 1 requires light energy, it is referred to as the light stage or light reactions. On the
other hand, stage 2 does not require light energy and is referred to as the dark stage or dark
reactions.
Within plants, the whole process occurs within the intracellular organelle, the chloroplast which is
believed to have bacterial origins.
stroma or cytoplasm
thylakoid vesicles
4-4
All of the reactions to do with the light stage (absorption of light energy and oxidation of water)
occur in the membrane of the thylakoid vesicles and all of the reactions to do with the dark stage
(CO2 reduction to carbohydrate) occur in the stroma.
2. Light Reactions
The principal light absorbing molecule is the chlorphyll of which there are several different types
varying in substituents on the porphyrin ring. There are light harvesting complexes composed of
many proteins, chlorophylls and other pigments that absorb light energy and transfer it to one of
two complex apparatuses called photosystems I and II (PS I and PS II). The complexity of the
photosystems is evident in photosystem I which contains 16 proteins, 168 chlorphylls, as well as
carotenoids, Fe-S clusters and phylloquinones.
CH2CH3
H3C
N
N
This is the structure of a chlorophyll
but you are NOT responsible for it.
The point here is to illustrate the
conjugated double bond system.
Mg
N
N
CH3
H3C
C20H39OOCH2CH2C
phytol
H3COOC
O
To understand how light energy is absorbed, we must first briefly review what light energy is.
Light energy has the properties of both a particle or photon and a wave. The speed of light is
expressed by:
c = λν
(where λ is the wavelength and ν is the frequency.)
The energy of light is expressed by:
E = hν
or
E = hc/λ
(where h is Planck's constant)
It is more common to use wavelength than frequency as a measure and the energy of an Einstein
(6 x 1023) of photons increases with decreasing wavelength.
4-5
red
purple
λ
650
590
490
395
kJ/mol
181.8
201.0
242.0
300.1
The important point here is that the energy of a photon
of light increases as you move to shorter wavelength.
This is an important consideration for exposure to
sunlight.
UV
All electrons in a molecule have the capability to exist in a number of different energy levels or
energy states. The most stable is the ground state, but absorption of energy can result in an
electron being excited to a higher energy level. Light energy can be used for this photoexcitation.
E3
E2
hν must equal the energy required for excitation from one
level to another.
E1
The energies of the transitions vary with the type of electron
and π electrons in an extended aromatic system are not as
tightly bound and can be excited at lower energy levels
whereas those more tightly bound (n electrons) require
higher energy.
hν
(purple)
hν
(red)
E0
Absorbance
The absorbance spectrum of a chlorophyll
exhibits two main peaks of absorbance, one
in the red region (650 to 700 nm) and the
second in the blue region (400 nm) of the
spectrum. The trough between the two
peaks where light is not absorbed falls in the
green region of the spectrum where light is
reflected giving chlorophyll and plants their
green color.
300
400
500
600
700
Wavelength (nm)
The light reactions actually involve two key photoexcitations involving photosystems I and II once.
In fact, some of the light energy is transferred in from the light harvesting complexes but the
photosystems themselves have a very complex array of chlorophylls and pigments that can absorb
light. The light energy is eventually transferred to a key chlorophyll or reaction center in each of
the photosystems, P700 in PS I and P680 in PS II (denoted by the wavelength of maximum
absorbance).
hν
P680
P680* (in PSII)
o
o
E' = -0.6 v
E' = +1.0 v
hν
P700
P700* (in PSI)
o
o
E' = +0.4 v
E' = -1.0 v
4-6
The significance of an electron photoexcited to a higher energy level is that it is less tightly bound to
the molecule and can therefore be donated or given up to another molecule more easily. In other
words, the molecule becomes a better reducing agent or is more easily oxidized.
This is evident in the change in reduction potentials from +1.0 v to -0.6 v and +0.4 v to -1.0v. When
compared to the standard reduction potential of NADP+ of -0.32 v, it means that the photoexcitation
of the reaction center can lead to reduction of NADP+.
-1.0v
Effectively, electrons are pumped up
hill or against an energy barrier
using light energy.
NADP+
NADH
Eo'
energy out
as ATP O2
light energy in
H2O
O2
+1.0v
-1.0v
P700*
P680*
Eo'
Two key photoexcitations in PS II
and PS I are used to bring about the
electron transfer from H2O to
NADP+.
NADP+
ATP
hν
PSI
0.0v
hν
PSII
P700
O2
H2O
OEC
P680
+1.0v
This diagram is often referred to as the "Z" diagram of the light reactions of photosynthesis and its
basic tenet is that it delineates (1) how electrons are photoexcited from water to NADP+, (2) that O2
is evolved and (3) that ATP is generated as a result of electron flow (the mechanism will come
shortly).
PS I
see p 4-9
-1.0v
P700*
PS II
Fe-S protein
P680*
Fe-S protein
ferridoxin
ferridoxin
pheophytin
hν
Eo'
4-7
Lec #12
NADP+
plastoquinone
cytochrome bf
0.0v
hν
plastocyanin
cytochrome bfquinone
complex
P700
OEC
H2O
+1.0v
2H+
1/2O2
P680
The OEC or oxygen evolving complex is a manganese containing complex that is responsible for
pulling electrons out of water and generating molecular oxygen.
The series of electron transfer reactions linking P680* and P700 are similar in concept to the
series of oxidation-reduction reactions that make up the Electron Transport Chain in the
membranes of mitochondria and bacteria. The similarity extends further to there being protons
pumped across the thylakoid membrane to generate a pH and electrical gradient. This gradient
or energized state is then used by ATPase to generate ATP.
Getting a little bit ahead of ourselves, 2 NADPH and 3 ATP are required to fix 1 CO2 into
organic form and the quantum yield refers to the number of photons required to fix 1 CO2 or the
number of photons required to generate 2 NADPH and 3 ATP.
From what we have seen so far, determining the number of photons required to generate 2
NADPH is clear.
8 hν
2 H2O
O2 + 2 H+
4 electrons are involved and each electron
must be photoexcited 2 times: 2 x 4 = 8 or
+
2 NADP
2 NADPH
it takes 8 photons to generate 2 NADPH.
4-8
The next question is how is ATP generated and how many photons are required to generate 3
ATP? The answer lies in the chemiosmotic theory, first introduced for oxidative phosphorylation in
the mitochondria. As the electrons flow through the system, protons are pumped across the
membrane INTO the thylakoid vesicle. This creates a region of positively charged electrical
character and low pH inside the vesicle and a region of negatively charged electrical character and
high pH in the stroma. In other words, an energized state is created which can be dissipated by the
flow of protons across the membrane. 4 protons are generated at the OEC and another 8
protons are pumped at the cytochrome bf / quinone complex for a total of 12 H+ per 4 electrons.
As in the mitochondria, there is an ATPase in the thylakoid membrane through which protons can
flow and the released energy is coupled to the phosphorylation of ADP to form ATP. The yield is
also similar with 4 H+ yielding 1 ATP.
In response to 8 photons:
1.
2 H2O + 2 NADP+
O2 + 2 NADPH + 2 H+ and,
2.
12 H+ are pumped across the membrane and,
3.
3 ADP + 3 Pi
3 ATP + 3 H2O
as protons flow back through the ATPase.
This gives rise to a quantum yield of 8 photons per 2 NADPH and 3 ATP or 8 photons per 1 CO2
fixed into organic form.
Stroma (outside)
12 H+
4hν
3 ADP + 3 Pi
3 ATP + 3 H2O
4hν
2 NADP+ + 2 H+
2 NADPH
OEC
2 H2O
+
-
(1 H per e )
PS II
O2 +
4 H+
Cyt bf
PS I
(high pH)
-----------
ATPase
++++++++++++++
(low pH)
8 H+
(2 H+ per e-)
Thylakoid vesicle (inside)
4-9
The Z-diagram as just presented depicts a process of non-cyclic electron flow during which
electrons are pumped from H2O to form NADPH. There also exists a modification to this system
that allows a process of cyclic electron flow that seems to have evolved as a means to generate
additional ATP.
The system is a hybrid of PS I and the plastoquinone/cytochrome bf complex that bypasses
NADPH formation.
PS I
Electrons are photoexcited in PS I and
as they flow through the plastoquinone
and cytochrome bf complexes, protons
are pumped across the thylakoid
membrane. This generates the pH
gradient to be used by the ATPase
to generate ATP.
P700*
Fe-S protein
ferridoxin
hν
plastoquinone
cytochrome bf
cytochrome bf
quinone
complex
plastocyanin
P700
The locations or distribution of ATPase, PS I, PS II and the cytochrome bf - quinone complex differ
in the thylokoid vesicles. The ATPase and PS I are found mainly in the unstacked regions giving
them access to NADP+ and ADP in the stroma. PS II is found mainly in the regions of stacked
lamellae while the cytochrom bf - quinone complex is spread evenly throughout the membrane.
ATPase
ATPase
PS I
PS II
PS II
Cyt bf
Cyt bf
PS I
4-10
3. Dark Reactions
The dark stage or dark ractions are responsible for the fixation of CO2 into organic form and are
collectively known as the Calvin Cycle.
3 C5 + 3CO2
[ 3 C6]
6 C3
5 C3
1 C3 (profit)
As presented at this lowest common denominator of intermediates, the process requires 6
NADPH and 9 ATP (3 CO2 are fixed each requiring 2 NADPH and 3ATP).
To generate one hexose (glucose), this process would occur twice generating 2 C3 which would
be combined into a C6, and the energy requirement would be 12 NADPH and 18 ATP.
The dark reactons can be sub-divided into two stages; 1. the fixation stage and 2. the
rearrangement stage. In neither stage is light energy used, and only in the fixation stage are the
NADPH and ATP used.
fixation stage
3 C5 + 3CO2
6 C3
6 NADP+ + 9 ADP
6 NADPH + 9ATP
rearrangement stage
3 C5
5 C3
A. Fixation stage
1
2
H 2C
C
HC
HC
H 2C
OH
ATP
O
ADP
OH
OH
2
CH2OPO3
Ribulose-5-phosphate
Ribulose phosphate
kinase
C
OPO3
O
HC
OH
HC
OH
2
CH2OPO3
Ribulose-1,5-bisphosphate
4-11
2
C5 + CO2
[C6]
2 C3
2
2
H 2C
2
OPO3
C
H 2C
O
HC
OH
HC
OH
2
OPO3
C
OH
C
OH
HC
OH
2
H 2C
OOC
OPO3
C
OH
C
O
CO2
HC
CH
OH
O
C
H2O
OH
HC
OH
2
CH2OPO3
Ribulose-1,5bisphosphate
OOC
OPO3
O
2
CH2OPO3
H 2C
2
CH2OPO3
CH2OPO3
reaction intermediates
2X
3- phosphoglycerate
Ribulose bisphosphate
carboxylase
Ribulose bisphosphate carboxylase is a multimer of 8 large and 8 small subunits, L8S8 and is
probably the most abundant protein in nature.
3
2
O
O
O
C
2 ATP
OH
HC
2
CH2OPO3
OPO3
C
2 ADP
HC
3-Phosphoglycerate
kinase
OH
2
CH2OPO3
2X
1,3-bisphosphoglycerate
2X
3- phosphoglycerate
4
2
O
OPO3
2 NADPH + 2 H
C
HC
OH
2
O
+
2 NADP
+
H
CH2OH
C
HC
2
2 Pi
CH2OPO3
Glyceraldehyde-3-phosphate
dehydrogenase
2X
2X
glyceraldehyde1,3-bisphosphoglycerate
3-phosphate
CH2OPO3
C
OH
Triose phosphate
isomerase
O
2
CH2OPO3
1X
dihydroxy
acetone
phosphate
4-12
In summary, for the fixation stage reaction
OR
1 CO2 + 1 C5
2 C3,
3 CO2 + 3 C5
6 C3
Lec #13
3 ATP and 2 NADPH are required.
5 C3
1 C3 (profit)
B. Rearrangement stage
The overall reaction is 5 C3
3 C5 and much of the process should be considered along side the
pentose phosphate pathway. Like the pentose phosphate pathway, the easiest way to keep things
straight is to have an overall scheme that illustrates duplicated processes and then fit the details
into the overall template.
C3
1
Steps 1 and 3 utilize almost
the same enzymes, and steps
2 and 4 also utilize a second
enzyme.
C6
C3
2
C5 + C4
C3
3
C7
C3
4
C5 + C5
C3
OR
5 C3
3 C5
1
C3 + C3
O
H
2
CH2OH
C
HC
C6
CH2OPO3
CH2OH
C
C
O
H2O
OH
+
C
O
HO
2
2
CH2OPO3
glyceraldehyde3-phosphate
CH2OPO3
dihydroxy
acetone
phosphate
Aldolase
Pi
HO
CH
HC
OH
HC
OH
2
CH2OPO3
fructose-1,6bisphosphate
O
Fructose, 1,6bisphosphatase
CH
HC
OH
HC
OH
2
CH2OPO3
fructose-6phosphate
4-13
2
C6 + C3
C4 + C5
CH2OH
CH2OH
C
HO
O
O
HC
H
C
C
CH
+
HC
Transketolase
OH
HC
TPP
2
HC
HC
HO
O
CH
OH
CH2OPO3
OH
+
OH
HC
OH
O
HC
2
OH
CH2OPO3
glyceraldehyde3-phosphate
2
CH2OPO3
2
CH2OPO3
erythrose-4phosphate
fructose-6phosphate
xylulose5-phosphate
2
3
C4 + C3
HC
O
HC
OH
HC
C7
CH2OPO3
CH2OH
C
C
O
CH2OH
HO
C
dihydroxy
acetone
phosphate
erythrose-4phosphate
HC
OH
HC
OH
Sedoheptulose-1,7bisphosphatase
HC
OH
HC
OH
HC
OH
Aldolase
CH2OPO3
CH2OPO3
OH
HC
2
2
CH
O
+
OH
Pi HO
H2O
CH
2
2
CH2OPO3
CH2OPO3
4
O
C7 + C3
sedoheptulose1,7-bisphosphate
C5 + C5
sedoheptulose7-phosphate
CH2OH
C
HO
HC
O
CH2OH
HC
OH
C
Transketolase HC
OH
HC
OH
O
O
CH
H
C
HC
OH
HC
OH
HC
OH
2
+
HC
OH
2
CH2OPO3
glyceraldehyde3-phosphate
CH2OPO3
sedoheptulose-7-phosphate
TPP
+
2
CH2OPO3
ribose5-phosphate
HO
O
CH
HC
OH
2
CH2OPO3
xylulose5-phosphate
4-14
To finish up reactions 2 and 4, it is necessary to convert the 2 xylulose-5-P and 1 ribose-5-P to
ribulose-5-P and this is accomplished as follows:
HC
O
CH2OH
CH2OH
HC
OH
C
C
HC
OH
HC
OH
HC
OH
HC
OH
Ribose phosphate
isomerase
2
O
HO
Ribulose phosphate
3-epimerase
O
CH
HC
OH
2
2
CH2OPO3
CH2OPO3
CH2OPO3
1X
ribose5-phosphate
ribulose5-phosphate
2X
xylulose5-phosphate
In summary the overall process of fixation and rearrangement is:
6 NADPH + 9ATP
fixation
3 C5 + 3 CO2
6 C3
rearrangement
5 C3
1 C3 (profit)
5. C4 plants
The process just described occurs in all plants and produces a C3 carbohydrate (3phosphoglycerate) as the immediate product of CO2 fixation.
Some plants have evolved an accessory system that results in a C4 carbohydrate being the
immediate product of CO2 fixation. Such plants are referred to as C4-plants and the primary
reason for the auxiliary pathway is to allow the plants to grow more efficiently at lower CO2
concentrations. That is, C3 plants express only the Calvin Cycle process while C4 plants express
both the Calvin Cycle enzymes and the enzymes of the C4 process.
The reason that the C4 process may have evolved is that there is an inherent inefficiency in
ribulose bisphosphate carboxylase in the form of a side reaction which leads to the oxidation of
ribulose bisphosphate, rather than its carboxylation, and subsequent cleavage of the product to 2phosphoglycolate and 3-phosphoglycerate. In other words, the enzyme uses up both oxygen
and carbohydrate.
4-15
2
CH2OPO3
2
H 2C
OPO3
C
O
O
HC
OH
HC
+
2 - phosphoglycolate
(salavageable but
expensive)
O
O2
Ribulose bisphosphate
carboxylase/oxygenase
OH
2
O
O
C
3 - phosphoglycerate
HC
CH2OPO3
OH
2
Ribulose-1,5-bisphosphate
CH2OPO3
The problem for the enzyme lies in its relative affinities for O2 and CO2 in comparison to the
aqueous concentrations of the two compounds.
for O2 KM = 350 M compared to the aqueous [O2] of 250 M
This means the enzyme will work as an oxidase at less than 1/2 Vmax.
for CO2 KM = 9 M compared to the aqueous [CO2] of 10 M
This means the enzyme will work as a carboxylase at about 1/2 Vmax.
Or in other words, under normal conditions, the enzyme is a reasonably efficient oxidase as well
as a carboxylase. "Product pull" caused by the presence of NADPH and ATP generated in the
light reactions during day time creates a favorable environment for the other reactions of the Calvin
Cycle and pushes the enzyme to be a carboxylase. However, at night, in the absence of NADPH
and ATP, the enzyme works effectively as an oxidase and some estimates have has much as 50%
of fixed carbon actually being metabolized as a result.
C4 plants have evolved a system that circumvents this problem by creating an effective "CO2
pump" that increases the intracellular [CO2] available for ribulose bisphosphate carboxylase. The
system involves four additional enzymes and an extra cell type. We will look at the enzymes
individually and then consider the overall picture.
1
(HCO3-)
CO2
CO2
CO2
Pi
2
C
CH2
PEP
C
OPO3
PEP carboxylase
G'o=-28.6 kJ/mol
H 2C
CO2
OAA
O
4-16
2
CO2
O
C
H
OH
C
Malate dehydrogenase
∆G'o=-29.7 kJ/mol
CH2
3
NADP+
NADPH + H+
CO2
CH2
CO2
CO2
OAA
Malate
CO2
HC
NADP+
NADPH + H+
CO2
OH
C
H 2C
O
Malic enzyme
CH3
∆G'o=+1.7 kJ/mol
CO2
Malate
H2O
4
ATP
CO2
to ribulose
bisphosphate
carboxylase
Pyruvate
CO2
AMP + PPi
IPPase
2 Pi
CO2
2 ATP
equivalents
2
C
O
+ Pi
CH3
C
Pyruvate
orthophosphate
dikinase
Pyruvate
OPO3
CH2
PEP
In this case, it is the inorganic pyrophosphatase that pulls the reaction
towards PEP generating an overall ∆G'o for both reactions of ~0.
CO2 (atmosphere)
The process occurs in two
different cell types and
results in the CO2 being
pumped into the bundle
sheath cell creating an
elevated intracellular
concentration of CO2 for
use by ribulose bisphosphate
carboxylase.
OAA
PEP
malate
pyruvate
malate
pyruvate
Mesophyll cell
Bundle sheath cell
CO2
Calvin Cycle
4-17
This system provides better reaction conditions for ribulose bisphosphate carboxylase in the form of
a higher [CO2] making possible a more efficient fixation of CO2 and a more rapid accumulation of
organic carbon with less oxidation reaction.
Generally, C3 plants are found in temperate regions and C4 plants are found in the tropics, but there
are obvious exceptions. Rapidly growing plants such as crab grass and corn are C4 plants and the
growth advantage provided by the C4 process is obvious.
The C4 process requires more energy but with the benefit of faster growth.
C3 plants
12 NADPH + 12 H+
12 NADP+
6 CO2
(CH2O)6 + 6 H20
18 ATP + 18 H2O
C4 plants
12NADPH + 12 H+
18 ADP + 18 Pi
12 NADP+
6 CO2
(CH2O)6 + 6 H2O
30 ATP + 30 H2O
30 ADP + 30 Pi
In other words, an additional 2 ATP per CO2 are required for the C4 process to proceed.
6. Carbohydrate from acetate (AcCoA)
From section 1, 2 and 3 involving the degradation of various substrates to form acetyl CoA, one
take home lesson should have been that the only thing that can happen to acetate (a 2 carbon
molecule) is that it can be fed into the TCA cycle to generate 2 CO2. In other words, there can
be no net gain in organic carbon atoms using acetate as a carbon source for growth
acetyl CoA
(C2)
(citrate)
(OAA)
CO2
CO2
4-18
However, there are many obvious examples in nature where acetate can be used as a carbon
source from which larger organic molecules are generated. The most obvious lies in plants during
seed germination when lipids in the seeds are broken down to acetate (β-oxidation) and used to
generate rootlets and stems. Also, bacteria can grow using acetate as the sole carbon source.
In order to do this, it is necessary to bypass the two decarboxylation steps in the TCA cycle and this
is achieved by the glyoxalate shunt. This involves two additional enzymes superimposed on the
TCA cycle.
TCA cycle
CO2
CO2
CH2
CH2
H 2C
H
C
HC
CO2
CO2
succinate
CO2
Isocitrate lyase
OH
acetyl-CoA
HC
CH2
CoASH
O
HO
CO2
CO2
Malate synthase
glyoxalate
Isocitrate
C
CO2
Malate
Ac-CoA
1
citrate
isocitrate
OAA
CO2
2
α-KG
glyoxalate
malate
succinyl-CoA
CO2
fumarate
sucinate
The difference between the TCA cycle and the glyoxalate shunt is as follows:
OAA + 2 AcCoA
OAA + 2 AcCoA
2 OAA (glyoxalate shunt)
OAA + 4 CO2 (TCA cycle)
bypassed
reactions
H
Glyoxalate enzymes
4-19
Lec #14
As noted earlier, plants utilize the glyoxalate shunt during seed germination which is a very specific
growth stage. As a result the glyoxalate enzymes are synthesized for only a short length of time as
they are needed and then synthesis is turned off. This is illustrated in the graph.
seed
plant
germination
Time
Another way of looking at the process to reinforce its role is as follows:
AcCoA
(C2)
Isocitrate
(C6)
AcCoA + OAA
(C2)
(C4)
2 OAA (net gain of 4 C via glyoxalate)
(2C4)
2 CO2
OAA (no net gain of C in TCA cycle)
(1C4)
7. Synthesis and storage of glucose
Once photosynthesis produces the glyceraldehyde-3-P and the glyoxalate shunt produces OAA, the
products can be converted into glucose via the gluconeogenesis pathway which is basically the
reversal of glycolysis with a couple extra enzymes added. This was covered in detail in section 1 of
the notes.
Pyr
OAA
PEP
2-PGA
3-PGA
1,3-bisPGA
Ga-3P
Frc-1,6-bisP
Frc-6-P
Glc-6-P
Excess glucose produced in this way can then be stored as glycogen:
glc-6-P
glc-1-P
glycogen
The following section considers the reactions involved in glycogen synthesis and breakdown and
then looks at the more interesting (and complicated) regulatory system that controls the process.
4-20
2
O3POH2C
HOH2C
O
HO
O
HO
HO
HO
Phosphoglucose
mutase
OH
OH
2
OH
OPO3
glucose-6-phosphate
glucose-1-phosphate
O
HOH2C
NH
O
HO
HO
N
OH
O
OPO2OPO2O
HOH2C
O
HO
UDPG pyrophosphorylase
O
HO
OH
PPi
UTP
2
IPPase
H2O
H
OH
H
OH
H
OPO3
glucose-1-phosphate
H
uridine diphosphoglucose
(UDPG)
2 Pi
HOH2C
O
HO
HO
HOH2C
O
O
OH
Glycogen synthase
(GS)
HO
OH
n glc
glycogenn+1
UDP
HOH2C
O
HO
HO
HOH2C
OH
O
O
HO
HOH2C
added to end of chain last
removed from chain first
last in - first out
OH
glycogenn+2
O
O
HO
OH
n glc
4-21
HOH2C
O
HO
HO
HOH2C
OH
O
O
HO
HOH2C
OH
Pi
O
O
HO
OH
glycogenn+2
Glycogen phosphorylase
(GP)
n glc
HOH2C
HOH2C
O
HO
O
HO
HO
HO
HOH2C
OH
OH
O
O
2
OPO3
HO
glucose-1-phosphate
OH
glycogenn+1
n glc
Overall
GP
Pi
gluconeogenesis
glc-6-P
glycolysis
glc-1-P
glycogenn+1
glycogenn+2
GS
UTP
UDP
UDPG
PPi
2 Pi
In the absence of a system to control these reactions, there is the potential for a "futile cycle"
which would use up UTP (equivalent to ATP in energy terms). Therefore, glycogen metabolism
is highly regulated with hormones affecting the activity and synthesis of the enzymes.
4-22
8. Regulation of glycogen metabolism
While there are several levels of control of glycogen metabolism, the best studied and
understood is the one activated by adrenalin (epinephrin or norepinephrin) secreted in the
adrenal cortex in response to some external stimulus. The hormone binds to a β-adrenergic cell
surface receptor present on certain tissue types. Closely associated with the receptor is the
enzyme adenylate cyclase and whatever change in the receptor is induced by adrenalin binding
results in activation of the cyclase and an increase in cAMP levels.
NH2
N
O
-O
P
O
O
O-
P
N
O
O
P
O-
O
N
2 Pi
H2O
H
N
IPPase
PPi
N
N
O
O-
NH2
O
H
H
OH
H
OH
Adenylate cyclase
O
P
H
H
O
H
OH
H
O-
cAMP
H2O
The phosphodiesterase is active at a low level at
all times (except when inhibited by caffeine) and
the levels of cAMP are determined by the activity of
the cyclase and whether it is turned on or not.
Phosphodiesterase
NH2
Therefore, levels of cAMP rise when it is turned on
and drop when it is turned off as a result of the
slow action of the phosphodiesterase.
N
N
O
cAMP is an intracellular messenger with roles in
many different processes. In the case of glycogen
metabolism regulation, it interacts with the
regulatory subunit (R) of protein kinase (PK)
causing its dissociation from the catalytic subunit
(C) which is activated as a result.
cAMP
-O
P
O
N
N
O
OH
H
H
OH
H
OH
5'-AMP
protein
R
Pi
cAMP
R
N
O
ATP
C
N
ATP
C
ADP
Protein
phosphatase
H2O
inactive
protein-P
active
Any protein that is phosphorylated has to be dephosphorylated and this is accomplished by protein
phosphatase (PP).
4-23
Once activated the protein kinase phosphorylates three proteins that affect glycogen metabolism.
1. Glycogen synthase (GS) is inactivated.
Protein kinase
GS
GS-P
(active)
(inactive)
ATP
ADP
This results in the turning off of glycogen synthesis.
2. Glycogen phosphorylase kinase (GPK) is activated to phosphorylate and activate glycogen
phosphorylase (GP).
Protein kinase
GPK
GPK-P
(inactive)
(active)
ATP
ADP
GPK-P
GP
(inactive)
ATP
GP-P
(active)
ADP
This results in the turning on of glycogen degradation.
3. Protein phosphatase inhibitor (PPI) is activated so that it can bind to and inactivate protein
phosphatase (PP).
Protein kinase
PPI
PPI-P
(inactive)
(active)
ATP
ADP
PP
PP-PPI-P
(active)
(inactive)
This results in the phosphorylation reactions not being reversed.
The net effect of these three reactions is that glycogen synthesis is stopped, glycogen breakdown
is turned on and the enzyme that reverses the effect of protein kinase, protein phosphatase, is
turned off.
This is summarized in the two diagrams on the following page. The top diagram shows how the
presence of adrenalin activates energy release and the bottom diagram shows how in the absence
of adrenalin, the system is shifted to carry out energy storage.
4-24
energy storage
Adrenalin
X
glycogenn+2
-adrenergic
receptor
adenylate
cyclase
X
GS
active
GP-P
active
glycogenn+1
X
X
PK
inactive
cAMP
GS-P
inactive
X
GP
inactive
energy release
GPK-P
active
PK
active
X
PPI
inactive
PP
active
GPK
inactive
X
PP - PPI-P
inactive
PPI-P
active
Adrenalin
energy storage
-adrenergic
receptor
glycogenn+2
X
GS
active
adenylate
cyclase
cAMP
5'AMP
PK
inactive
glycogenn+1
X
X
X
GS-P
inactive
energy release
PK
active
GP-P
active
X
GP
inactive
GPK-P
active
PPI
inactive
X
GPK
inactive
X
PPI-P
active
PP
active
PP - PPI-P
inactive
4-25
Lec #15
9. Summary of carbohydrate synthesis
1. Photosynthesis
a) overall reactions
b) light reactions - Z-diagram, NADPH and ATP synthesis
c) dark reactions - CO2 fixation
d) C4 reactions
2. Growth on acetate - glyoxalate shunt
3. Glycogen metabolism
a) review of gluconeogenesis
b) synthesis and degradation
c) regulation