Chapter 20:The Calvin Cycle and the

Pentose Phosphate Pathway

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Photosynthesis Dark Reactions (The Calvin cycle)

Reductive conversion of CO2 into carbohydrate. This process fixes ~1010 tons of CO2 annually.

Process is powered by ATP and NADPH which are products of the light reactions of photosynthesis.

Dark reactions occur in chloroplast stroma.

Called the Calvin-Benson-Bassham pathway or the reductive pentosephosphate cycle (RPP)

Net equation for the Calvin cycle

3 CO2 + 9 ATP + 6 NADPH + 5 H2O

9 ADP + 8 Pi + 6 NADP+ + *Triose phosphate

From an energy standpoint this is an expensive process: 3 ATP and 2 NADPH per CO2 incorporated.

*(G3P or DHAP)

These reactions occur in the stroma.

Calvin Cycle

Fixation,Reduction,Regeneration

Stage 1: Incorporation of CO2

Catalyzed by the enzyme Rubisco.Ribulose-1,5-bisphosphate carboxylase-oxygenase

Plants fed CO2, yield 3-phosphoglycerate as the first compound detected.

Rubisco

8 subunits @ 530008 subunits @ 14000~540000 d total

It is the most abundant enzyme in the biosphere

Rubisco Mechanism

Mg++ binding

to Rubisco

Bound using Glu, Asp and a Lys carbamate. Thecarbamate formation is catalyzed by Rubisco Activase

Stage 2

Phosphorylation and

Reduction

Hexose synthesis

3-phospho glycerate to hexose-P

Stage 3: Regeneration

These reactions serve to regenerate ribulose-1,5-bisphosphate from glyceraldehyde-3-phosphate.

Two group transfer reactions are common here:

1. a transketolase reaction using TPP and

2. a transaldolase reaction

Then an isomerase, an epimerase and a kinase complete the cycle.

Example Transketolase

2 C transfer from a ketose

TPP

Example Transaldolase

3 C transfer from a ketose

Sugar Interconversions

2 C transfer from ketose

TPP

Sugar Interconversions

3 C transfer from ketose

Sugar Interconversions

2 C transfer from ketose

TPP

Sugar Interconversions

Calvin Cycle

Enzymes of the Calvin Cycle

1. Rubisco 2. Phosphoglycerate kinase 3. Glyceraldehyde-3-phosphate dehydrogenase 4. Triosephosphate isomerase 5. Aldolase (transaldolase) 6. Fructose bisphosphatase 7. Transketolase 8. Sedoheptulose-7-phosphatase 9. Phosphopentose isomerase

10. Phosphopentose epimerase 11. Ribulose-5-phosphate

kinase

Carbon Flow in the Calvin Cycle

3 C5 + 3 C1 ---> 6 C3

2 C3 ---> 1 C6

C6 + C3 ---> C4 + C5

C4 + C3 ---> C7

C7 + C3 ---> 2 C5

-----------------------------------

net 3 C1 ---> 1 C3

Synthesis of Sucrose

Occurs in the cytosol.

There is a triose-P:Pi antiport in the chloroplast membrane.

Regulation of the Calvin Cycle

Rubisco has three forms:

1. E binds R-1,5-B in the dark and is inactive. R-1,5-B is an inhibitor in the dark (blocks the carbamylation site). Rubisco activase causes dissociation of R-1,5-B and catalyzes ATP dependent attachment of CO2.

2. EC carbamylated at Lys201 is still inactive.

3. ECM has bound Mg++ and is active.

Rubisco and rubisco activase are light activated.

Regulation of the Calvin Cycle

Rubisco activation requires: light, CO2, Mg++ & pH 7.4 (optimum is 8.1)

Functioning PSII-Cytbf-PSI cause proton pumping which leaves the stroma basic. The potential developed promotes translocation of Mg++ and Cl-. Mg++ is needed in the stroma for rubisco and both phosphatases. The stroma can reach pH 9.0. The high pH activates rubisco.

Light produces a conformational change in rubisco activase enhancing its activity.

Regulation of the Calvin Cycle

Functioning PSII-Cytbf-PSI also produces reduced ferredoxin and NADPH.

Ferredoxin-Thioredoxin Reductase generates thioredoxin-(SH)2

Ferredoxin(red) + thioredoxin-S2(ox) ===== > Ferredoxin(ox) + thioredoxin-(SH)2(red)

Thioredioxin (a small protein) activates a number of enzymes through a disulfide -- > dithiol conversion.

Regulation of Photosynthesis

Light is needed to generate NADPH, FDred and Mg++ transport

Thioredoxin

This is a small disulfide containing protein.

Action ofThioredoxin

Enzymes activated by thioredoxin:

Fructose bisphosphataseSedoheptulose bisphosphataseRibulose-5-P kinaseGlyceraldehyde-3-P dehydrogenase

Regulation

Photorespiration (Use of O2)

This competes with photosynthesis at higher temperatures so is typically more active in the summer and in the tropics.

Conc. in air KM

O2 250 μM (20%) 200 μM CO2 11 μM (0.04%) 20 μM

However, the affinity of Rubisco for CO2 decreases with increasing temperature.

The immediate products of photorespiration are phosphoglycolate and 3-phosphoglycerate.

Photorespiration

The mechanism is analogous to that for carboxylation. Rubisco must be carbamylated.

Photorespiration

The reaction involved in photorespiration require participation of enzymes from the cytosol, chloroplasts, mitochondria and peroxisomes.

Chloroplast: glycolate phosphatasePeroxisome: glycolate oxidase

transaminasehydroxypyruvate reductase

Mitochondria: Glycine cleavage enzyme serine hydroxymethyltransferase

Cytosol: glycerate kinase

Photo respiration reactions

Photo respiration reactions

Photorespiration

CHO

CO2-

H C OH

CH2 -OPO3=

C = O

CH2 -OPO3=

C = O

H C OH

H C OH

CH2 -OH

CH2 -OH

CO2-

CO2-

H C NH2

CH2 -OPO3=

CO2-

H C NH2

CH2 -OH

CO2-

H C OH

CH2 -OH

C = O

CH2 -OH

CO2-

CO2-

CH2 -OPO3=

CO2-

CH2 -OH

CO2-

CO2-

CH2 -NH2

2-P-glycolate glycolate glyoxylate

glycine

3-P-glycerate3-P-hydroxypyruvate 3-P-serine Serine

glycerate hydroxypyruvate

Ribulose-1,5-bisphosphate

Rubisco

+ O2

1 2 3

6

54

7 8

1. phosphoglycerate DH 5. glycerate dehydrogenase2. transaminase 6. glycerate kinase3. phosphoserine phosphatase 7. phosphoglycolate phosphatase4. transaminase 8. glycolate oxidase

PhotorespirationRun through the previous reactions twice yield two glycines which then react as shown below.So two glycolates produce 1 CO2 and 1 serine.

CO2-

CH2 -NH2

glycine

+ FH4 + NAD+ ---------- > CH2FH4 + NADH + NH4+

+ CO2

CO2-

CH2 -NH2

glycine

+ CH2FH4 ---------------- > FH4 +

CO2-

H C NH2

CH2 -OH

Serine

glycinecleavage

enzyme

serinehydroxymethyl

transferase

Tetrahydrofolate

FH is a coenzyme that serves as a one carbon carrier.

Hatch-Slack Pathway

This pathway uses a CO2 concentrating mechanism to permit photosynthesis to surpass photorespiration.

The initially observed compound is this case is oxaloacetate, so this is referred to as the C4 pathway. Similarly, normal photosynthesis is sometimes called the C3 pathway.

C4 plants include crabgrass, bermuda grass, corn, maize and sugarcane. These have an advantage over C3 plants in hot weather.

C4 Pathway

C4 plants have mesophyll cells which are outer cells that collect CO2. Bundle sheath cells are inside where the Calvin Cycle occurs.

C4 Pathway

CO2-

C OPO3= C = O

CO2-

CO2-

H C OH

PEP oxaloacetate malatepyruvate

1 2

3

1. PEP carboxylase 2. malate dehydrogenase3. Malic enzyme4. pyruvate:phosphate dikinase

CH2

C = O

CO2-

CH3

bundle sheath cell

mesophyll cell

CO2-

H C OH

4

CH2-CO2-

CH2-CO2- CH2-CO2

-

C = O

CO2-

CH3

CO2

CO2

to Calvin cycle

from air

Pi

NADPH

NADP+

NADPH

NADP+

ATP+Pi

ADP+2Pi

Hatch-Slack Pathway

Different plants have different mechanisms for moving CO2 into the bundle sheath cells. The Hatch- Slack uses four enzymes in the C4 pathway.

1. PEP carboxylase: HOH + PEP + CO2 -- > OAA + Pi2. Malate dehydrogenase (NADP+ dependent)3. Malic enzyme (NADP+ dependent) also called malate dehydrogenase decarboxylating 4. Pyruvate:phosphate dikinase

ATP + Pi -- > ADP + PPiADP + E -- > AMP + E~PE~P + Pyruvate -- > PEP + EPPi -- > 2 Pi

Hexose MonophosphateShunt (HMS)

Pentose phosphate pathway or Phosphogluconate pathway

This pathway is the major site for production of:1. NADPH for anabolism (reductive)2. Ribose-5-phosphate for nucleotide synth.

Other: Makes erythrose for Phe synthesis.

Completely oxidizes glucose without Krebs.No ATP used or made in this pathway.

Hexose monophosphate

shunt (HMS)

Phase 1 - oxidative.Phase 2 – isomerization, epimerization and rearrangement.

HMS

This phase is composed of three reactions, two of which are oxidations.

HMS

Isomerization and epimerizartion.

HMS

HMS – Oxidative Phase

The oxidative phase is one-way as written.Reaction 1 is the control site for the HMS. Reaction 3 is the least reversible step. The mechanism involves the decarboxylation of a -ketoacid, similar to isocitrate dehydrogenase.

HMS – 1st Oxidative Step

HMS – Lactone Formation

HMS – 2nd Oxidative Step

Sugar Interconversions

2 C transfer from ketose

TPP

Sugar Interconversions

3 C transfer from ketose

Sugar Interconversions

2 C transfer from ketose

TPP

Pentose Phosphate PathwayReactions

Transketolase Mechanism

Step 1

Step 2

Step 3

Step 4

Step 5

Transaldolase Mechanism

Step 1

Step 2

Step 3

Step 4

Step 5

Step 6

Transaldolase & TransaldolaseActive components of each mechanism

transketolase

transaldolase

Making Ribose-5-PEnter HMS from F-6-P and convert all to ribose-5-P

Making NADPH & Ribose-5-PEnter HMS from G-6-P, make NADPH and convert all ribulose-5-P to ribose-5-P

Making NADPHEnter HMS from G-6-P, convert all ribulose-5-P to fructose-6-P and recycle to glucose-6-P.

Making NADPH & ATPEnter HMS from G-6-P, convert all ribulose-5-P to fructose-6-P and use it for energy via glycolysis.

Active HMS

Regulation of the HMS

Glucose-6-phosphate dehydrogenase is the control point for the pathway. NADPH(-) competes for the binding site with NADP+. Also, fattyacyl CoA(-). Thus, regulation is tied to the need for anabolism and reductive processes. KM of the enzyme for NAD+ is 1000 times greater that that for NADP+.

Normal levels: NAD+/NADH = 700-1000 and a high level of

NAD favors oxidation reactions.

NADP+/NADPH = 0.01-0.014 and high levels of NADP favor reduction reactions.

Erythrocytes

The need for NADPH in red cells is critical. Red cells lack mitochondria therefore no energy is available from the Krebs cycle or ET/OP. All energy is derived from glycolysis and the HMS.

NADPH is needed to keep hemoglobin and other proteins in the active dithiol form. The active agent here is glutathione, a ubiquitous reducing agent (found in all cells). Glutathione is -glutamyl-cysteinylglycine (GSH).

Activation of an oxidized enzyme:

2 GSH + ES2 -- > GSSG + E(SH)2

Glutathione (Found in all cells.)

- carboxyl

thiol

Reduction of GSSG

Glutathione reductase is an NADPH requiring flavoprotein that catalyzes conversion of GSSG back to 2 GSH. FADH2 does not convert GSSG directly but rather goes through a disulfide/ dithiol conversion on the enzyme.

NADPH + H+ + E-FAD -- > NADP+ + E-FADH2

E-FADH2 + ES2 -- > E-FAD + E(SH)2

E(SH)2 + GSSG -- > ES2 + 2 GSH

The normal GSH/GSSG ratio in red cells is ~500.

End of Chapter 20

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition