4-1Carbohydrate 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 besupplied 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 organic carbon O2 respiration photosynthesis CO2energy released

(ATP)energy used (hν or light energy)

Lec # 11

nucleotides

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 casesto 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 (CH2O)6 + 12 CH3COCH3 + 6 H2O (isopropanol) (acetone) Comparison of the three overall reactions produces the generalized overall reaction:

6 CO2 + 12 H2X (CH2O)6 + 12 X + 6 H2O electron electron acceptor donor 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:

Stage 1 12 H2X 24 H+ + 24 e- + 12 X

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 H2O + 2 Fe3+ 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 (H2

18O) was used with the result that only 18O2 was found - no 16O2 (from the C16O2) or (CH2

18O)6. Then if the overall reaction is written normally, it is not balanced correctly.

6 C16O2 + 6 H218O (CH2

16O)6 + 618O2 However, it can be balanced simply by adding 6 H2

18O to the left and 6 H216O to the right side.

6 C16O2 + 12 H218O (CH2

16O)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. hνStage 1 12 H2

18O + 12 NADP+ 6 18O2 + 12 NADPH + 12 H+

Stage 2 6 C16O2 + 12 NADPH + 12 H+ (CH216O)6 + 6 H2

16O + 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

4-4All 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.

N

N N

N

Mg

H3C CH2CH3

CH3H3C

C20H39OOCH2CH2COH3COOCphytol

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.

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 λ kJ/mol red 650 181.8 590 201.0 490 242.0 purple 395 300.1 UV

The important point here is that the energy of a photonof light increases as you move to shorter wavelength. This is an important consideration for exposure to sunlight.

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.

E3E2

E1

E0

hν hν(red) (purple)

hν must equal the energy required for excitation from one level to another.

The energies of the transitions vary with the type of electronand 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.

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.

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 absorblight. 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) E'o = +1.0 v E'o = -0.6 v hν P700 P700* (in PSI) E'o = +0.4 v E'o = -1.0 v

300 400 500 600 700Wavelength (nm)

Abs

orba

nce

4-6

The significance of an electron photoexcited to a higher energy level is that it is less tightly bound tothe 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. Whencompared to the standard reduction potential of NADP+ of -0.32 v, it means that the photoexcitationof the reaction center can lead to reduction of NADP+.

Eo'

-1.0v

+1.0v

NADH

O2 H2O

NADP+

energy out as ATP

light energy in

Effectively, electrons are pumped uphill or against an energy barrier using light energy.

Two key photoexcitations in PS II and PS I are used to bring about theelectron transfer from H2O to NADP+.

H2OO2

P680

P680*

-1.0v

Eo'

+1.0v

0.0v

P700

P700*

hν

hν

OEC

PSII

PSIATP

NADP+

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).

O2

4-7

H2O1/2O2

P680

P680*

-1.0v

Eo'

+1.0v

0.0v

P700

P700*

hν

hν

OEC

PS II

PS I

NADP+

2H+

pheophytin

plastoquinone

cytochrome bf- quinone complex

plastocyanin

Fe-S protein

ferridoxin

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.

cytochrome bf

Lec #12

Fe-S protein

ferridoxin

see p 4-9

OEC PS II Cyt bf

4hν

4hν

8 H+

4-8

ATPase

12 H+

3 ATP + 3 H2O3 ADP + 3 Pi

2 NADP+ + 2 H+

2 NADPH

Stroma (outside)

Thylakoid vesicle (inside)

++++++++++++++

- - - - - - - - - - -

(low pH)

(high pH)

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 theflow 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 CO2fixed into organic form.

PS I

2 H2O O2 + 4 H+

(1 H+ per e-) (2 H+ per e-)

4-9

P700

P700*

hν

PS I

plastoquinone

cytochrome bf quinone complex

plastocyanin

Fe-S protein

ferridoxin

cytochrome bf

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.

Electrons are photoexcited in PS I and as they flow through the plastoquinoneand cytochrome bf complexes, protonsare pumped across the thylakoidmembrane. This generates the pHgradient to be used by the ATPase to generate ATP.

The locations or distribution of ATPase, PS I, PS II and the cytochrome bf - quinone complex differin 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 ATPasePS I

PS I

PS II

PS II Cyt bf

Cyt bf

4-103. 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 NADPH + 9ATP 6 NADP+ + 9 ADP rearrangement stage 5 C3 3 C5

A. Fixation stage

H2C OH

C O

HC OH

HC OH

CH2OPO32

Ribulose-5-phosphate

H2C OPO3

C O

HC OH

HC OH

CH2OPO32

Ribulose-1,5-bisphosphate

2

ATP ADP

Ribulose phosphate kinase

1

H2C OPO3

C O

HC OH

HC OH

CH2OPO32

Ribulose-1,5-bisphosphate

2

4-112

H2C OPO3

C OH

C OH

HC OH

CH2OPO32

2H2C OPO3

C OH

C O

HC OH

CH2OPO32

2

OOC

H2C OPO3

CH OH

2

OOC

C

HC OH

CH2OPO32

O O

CO2 H2O

reaction intermediates 2X3- phosphoglycerate

Ribulose bisphosphate carboxylase

C5 + CO2 [C6] 2 C3

Ribulose bisphosphate carboxylase is a multimer of 8 large and 8 small subunits, L8S8 and is probably the most abundant protein in nature.

C

HC OH

CH2OPO32

O O

2X3- phosphoglycerate

C

HC OH

CH2OPO32

O OPO3

2X1,3-bisphosphoglycerate

2

2 ATP 2 ADP

3-Phosphoglycerate kinase

C

HC OH

CH2OPO32

O OPO3

2X1,3-bisphosphoglycerate

2

3

C

HC OH

CH2OPO32

O H

2Xglyceraldehyde- 3-phosphate

CH2OH

C O

CH2OPO32

1Xdihydroxy acetone phosphate

4

2 NADPH + 2 H+ 2 NADP+

2 PiGlyceraldehyde-3-phosphate dehydrogenase

Triose phosphate isomerase

4-12In summary, for the fixation stage reaction 1 CO2 + 1 C5 2 C3, 3 ATP and 2 NADPH are required. OR 3 CO2 + 3 C5 6 C3 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 C6C3 2 C5 + C4C3 3 C7C3 4 C5 + C5C3 OR

5 C3 3 C5

Steps 1 and 3 utilize almost the same enzymes, and steps 2 and 4 also utilize a second enzyme.

C

HC OH

CH2OPO32

O H

glyceraldehyde- 3-phosphate

CH2OH

C O

CH2OPO32

dihydroxy acetone phosphate

Aldolase

C O

CHHO

HC OH

HC OH

CH2OPO32

fructose-1,6-bisphosphate

CH2OPO3

+

C O

CHHO

HC OH

HC OH

CH2OPO32

fructose-6-phosphate

CH2OH2

H2O Pi

Fructose, 1,6-bisphosphatase

1 C3 + C3 C6

Lec #13

4-132 C6 + C3 C4 + C5

C O

CHHO

HC OH

HC OH

CH2OPO32

fructose-6-phosphate

CH2OH

C

HC OH

CH2OPO32

O H

glyceraldehyde- 3-phosphate

+HC OH

HC OH

CH2OPO3

2

erythrose-4-phosphate

HC O

CH2OH

C O

CHHO

HC OH

CH2OPO32

xylulose-5-phosphate

HC O

HC OH

HC OH

HC OH

CH2OPO32

ribose-5-phosphate

+Transketolase

HC OH

HC OH

CH2OPO3

2

erythrose-4-phosphate

HC O

+

CH2OH

C O

CH2OPO32

dihydroxy acetone phosphate

CHHO

HC OH

HC OH

HC OH

CH2OPO32

sedoheptulose-1,7-bisphosphate

C

CH2OPO3

O

CHHO

HC OH

HC OH

HC OH

CH2OPO32

sedoheptulose-7-phosphate

C

CH2OH

O

2

Aldolase

H2O Pi

Sedoheptulose-1,7-bisphosphatase

3 C4 + C3 C7

C7 + C3 C5 + C54

CHHO

HC OH

HC OH

HC OH

CH2OPO32

sedoheptulose-7-phosphate

C

CH2OH

O

C

HC OH

CH2OPO32

O H

glyceraldehyde- 3-phosphate

+ Transketolase +

CH2OH

C O

CHHO

HC OH

CH2OPO32

xylulose-5-phosphate

TPP

TPP

4-14

CH2OH

C O

HC OH

HC OH

CH2OPO32

ribulose-5-phosphate

HC O

HC OH

HC OH

HC OH

CH2OPO32

1Xribose-5-phosphate

CH2OH

C O

CHHO

HC OH

CH2OPO32

2Xxylulose-5-phosphate

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:

Ribulose phosphate 3-epimerase

Ribose phosphate isomerase

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)

The process just described occurs in all plants and produces a C3 carbohydrate (3-phosphoglycerate) 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 expressboth 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 2-phosphoglycolate and 3-phosphoglycerate. In other words, the enzyme uses up both oxygen and carbohydrate.

5. C4 plants

4-15

H2C OPO3

C O

HC OH

HC OH

CH2OPO32

Ribulose-1,5-bisphosphate

2

C

HC OH

CH2OPO32

O O

3 - phosphoglycerate

+ O2

CH2OPO3

OO

2 - phosphoglycolate(salavageable but expensive)

2

Ribulose bisphosphatecarboxylase/oxygenase

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 wellas 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 CalvinCycle 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

PEP

CO2

C OPO3

CH2

2

PEP carboxylase

CO2

CO2

C O

H2C

CO2

Pi

OAA

G'o=-28.6 kJ/mol

(HCO3-)

4-16

Pyruvate

CO2

C O

CH3Malic enzyme

CO2

CO2

HC OH

H2C

CO2

Malate

NADPH + H+NADP+3

2

CH2

CO2

C

CO2

H OH

Malate

CH2

CO2

C

CO2

O

OAA

Malate dehydrogenase

∆G'o=+1.7 kJ/mol

NADP+NADPH + H+

Pyruvate

CO2

C O

CH3

4

PEP

CO2

C OPO3

CH2

2

Pyruvate orthophosphatedikinase

Pi+

ATP AMP + PPi 2 Pi

IPPase

to ribulose bisphosphate carboxylase

H2O

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)

OAA PEP

malate pyruvate

malate pyruvate

CO2 Calvin Cycle

Mesophyll cell

Bundle sheath cell

The process occurs in two different cell types and results in the CO2 beingpumped into the bundlesheath cell creating anelevated intracellularconcentration of CO2 for use by ribulose bisphosphatecarboxylase.

∆G'o=-29.7 kJ/mol

2 ATP equivalents

4-17This system provides better reaction conditions for ribulose bisphosphate carboxylase in the form ofa 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 thereare 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 18 ADP + 18 Pi C4 plants 12NADPH + 12 H+ 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 (citrate)

(C2) (OAA)

CO2 CO2

4-18However, 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 thisis achieved by the glyoxalate shunt. This involves two additional enzymes superimposed on the TCA cycle.

CO2CH

HC

CO2

CH2

CO2

Isocitrate

OH

CO2H2C

HC

CO2

CH2

CO2

glyoxalate

O

succinate

Isocitrate lyase CH2

CO2

C

CO2

HHO

Malate

Malate synthase

acetyl-CoA CoASH

TCA cycle

Ac-CoAcitrate

isocitrate

glyoxalate

sucinate

fumarate

malate

OAA

α-KG

succinyl-CoA

CO2

CO2

1

2

The difference between the TCA cycle and the glyoxalate shunt is as follows: OAA + 2 AcCoA 2 OAA (glyoxalate shunt)

OAA + 2 AcCoA OAA + 4 CO2 (TCA cycle)

bypassed reactions

4-19

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.

Another way of looking at the process to reinforce its role is as follows:

AcCoA 2 OAA (net gain of 4 C via glyoxalate) (C2) (2C4)AcCoA + OAA Isocitrate (C2) (C4) (C6) OAA (no net gain of C in TCA cycle) 2 CO2 (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 ofthe 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.

Time

Gly

oxal

ate

enzy

mes

seed germination plant

Lec #14

4-20

OO3POH2C

HOHO

OH OH

glucose-6-phosphate

2

OHOH2C

HOHO

OH

glucose-1-phosphate

2OPO3

Phosphoglucose mutase

OHOH2C

HOHO

OH

glucose-1-phosphate

2OPO3

OHOH2C

HOHO

OH

uridine diphosphoglucose (UDPG)

OPO2OPO2O

NH

O

ON

O

OHOH

HH

HHUTP PPi

2 Pi

UDPG pyrophosphorylase

IPPase

OHOH2C

HOHO

OHO

HOH2C

OHO

OH

OHOH2C

OHO

OHO

HOH2C

OHO

OH

OHOH2C

HOHO

OH

n glc

n glc

UDP

Glycogen synthase (GS)

added to end of chain lastremoved from chain first

last in - first out

glycogenn+1

glycogenn+2

H2O

4-21

OHOH2C

OHO

OHO

HOH2C

OHO

OH

OHOH2C

HOHO

OH

n glc

OHOH2C

HOHO

OHO

HOH2C

OHO

OH

n glc

OPO3

OHOH2C

HOHO

OH

Pi

Glycogen phosphorylase (GP)

glycogenn+1

glycogenn+2

glucose-1-phosphate

2

gluconeogenesis

glc-6-P glc-1-P glycolysis

UDPG

glycogenn+2glycogenn+1

Pi

UDPUTP

PPi

GP

GS

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.

Overall

2 Pi

4-228. 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 bindingresults in activation of the cyclase and an increase in cAMP levels.

N

NN

N

NH2

O

OHOH

HHHH

OPO

O-

O

PO

O-

O

P-O

O-

O

N

NN

N

NH2

O

OHO

HHHH

O

P

O-

OATP

cAMP

PPi

2 PiH2O

Adenylate cyclase

IPPase

N

NN

N

NH2

O

OHOH

HHHH

OP-O

O-

O

5'-AMP

H2O

Phosphodiesterase

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.

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.

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.

C R

R

C

cAMP

cAMP

inactive active

protein

protein-P

ATP

ADP

Pi

H2O

Proteinphosphatase

Any protein that is phosphorylated has to be dephosphorylated and this is accomplished by protein phosphatase (PP).

4-23Once 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 GP-P (inactive) (active) ATP 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 absenceof adrenalin, the system is shifted to carry out energy storage.

4-24Adrenalin

-adrenergicreceptor

adenylatecyclase

cAMP

PKinactive

PKactive

GSactive

GS-Pinactive

GPKinactive

GPK-P active

GP-Pactive

GPinactive

PPIinactive

PPI-Pactive

glycogenn+1

glycogenn+2

energy storage

energy release

PPactive

PP - PPI-P inactive

X

X

Adrenalin

-adrenergicreceptor

adenylatecyclase

cAMP

5'AMP

PKinactive

PKactive

GSactive

GS-Pinactive

GPKinactive

GPK-P active

GP-Pactive

GPinactive

PPIinactive

PPI-Pactive

glycogenn+1

glycogenn+2

energy storage

energy release

PPactive

PP - PPI-P inactive

X

X

X

X

X

X

X

X X

X

X

X

4-25

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

Lec #15