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Home , Phosphoglycerate kinase, Phosphoribulokinase, Pyruvate decarboxylase

BI/CH 422/622 ANABOLISM OUTLINE: Overview of Key experiments: Light causes oxygen, which is from water splitting (Hill) NADPH made (Ochoa) Separate from carbohydrate biosynthesis (Rubin & Kamen) Light Reactions energy in a photon pigments Carbon Assimilation – HOW Stage One – Rubisco Light absorbing complexes Carboxylase Reaction center Oxygenase Photosystems (PS) Glycolate cycle PSI – oxygen from water splitting Stage Two – making sugar PSII – NADPH Stage Three - remaking Ru 1,5P2 Proton Motive Force – ATP Overview and regulation Overview of light reactions Calvin cycle connections to biosyn. C4 versus C3 plants Kornberg cycle - glyoxylate

Photosynthesis Rubisco Carboxylase Mechanism― Overview Mg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolate intermediate

Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water

Recall another C-C cleavage of C3-tetrahedral a b-keto acid? intermediate

1 Photosynthesis Rubisco Carboxylase Mechanism― Overview Mg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolate intermediate

Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water

Recall another C-C cleavage of C3-tetrahedral a b-keto acid? intermediate

Photosynthesis First Stage of Calvin Cycle Oxygenase Activity of Rubisco • O2 competes with CO2 for the . ~1 in every 3 or 4 turnovers, O2 binds • The reactive in the reaction is the electron-rich enediol form of ribulose 1,5-bisphosphate.

• The nucleophile adds to O2 to form 3- phosphoglycerate (same as in Calvin cycle) and 2-phosphoglycolate (2-PG). –2-PG is metabolically difficult. –Salvaging its Carbons requires energy –As it is produced in appreciable amounts, an elaborate pathway has been cobbled together. This process is called PHOTO- RESPIRATION

2 Photosynthesis First Stage of Calvin Cycle 1 2 Dealing with the oxygenase activity of rubisco 1 The Glycolate Pathway* 2 1 • Complex ATP-consuming process for the 2

recovery of C2 fragments from photorespiration. 1st Glycolate is made. 1 2 • Uses three organelles • Loss of C as CO by mitochondrial 1 2 2 decarboxylation of glycine (see Pyr family)

• Two 2-PGs are converted to Ser + CO2. • The Ser is cycled back to the to 1 1 N5N10CH2 generate one 3-PGA. Serine OHMe THF 1 • Whole cycle costs an ATP per 1 3-PGA decarboxylase* converted back

*Don’t confuse with the glyOXYlate Cycle *called Glycine Cleavage in bacteria

Photosynthesis First Stage of Calvin Cycle Regulation of rubisco activity Rubisco is Inhibited by Several Criteria: 1. pH • In the stroma the pH is ~8 when light is on • pH decreases in the dark; rubisco inactive at low pH 2. CO2 (for the carbamoylation of Lys-210; also better at higher pH) 3. NADPH (indirectly through rubisco activase) 4. a “Noctural” Inhibitor • 2-carboxyarabinitol 1- inhibits carbamoylated Rubisco. – transition state analog of b-keto acid intermediate – synthesized in the dark in some plants

3 Photosynthesis The Three Stages of the Calvin Cycle of

10 CO2 Assimilation (6)

(6) (6)

Rubisco (10) (6) (2)

(12) (12)

(12) chloroplast isoforms (12) 3-phosphoglycerate (3PGK) (12) – (12) – glyceraldehyde 3-phosphate (12) dehydrogenase (GAPDH)

Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP → 2 glyceraldehyde 3- + + phosphate (GA3P) + 4 H + 12 NADP + 18 ADP + 16 Pi *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo)

Photosynthesis

Second Stage of Calvin Cycle: making 3-PGA Reduced to GA3P Twelve 3-phosphoglycerate + 12 ATP + 12 NADPH + 12 H+ à 12 + glyceraldehyde 3-phosphate + 12 ADP + 12 NADP + 12 Pi • Requires most of the ATP and NADPH from photosynthesis at ratio of 12:12 • Reverse of the key reactions in (except NADPH used rather than NADH) – uses chloroplast of 3- (3-PGK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) • Driven forward by high concentration of NADPH and ATP in the chloroplast stroma; and the pulling of GA3P and Pi by the cycle and ATP synthase

+ + ATP ADP NADPH+H NADP

Glyceraldehyde 3-Phosphoglycerate 1,3-Bisphosphoglycerate 3-phosphate

DG°’ = +5.5 kcal/mol –1.8 kcal/mol +3.7 kcal/mol

4 Photosynthesis The Three Stages of the Calvin Cycle of

10 enzymes CO2 Assimilation (6)

(6) (6)

Rubisco (10) (6) (2)

(12) (12)

(12) chloroplast isoforms (12) – 3-phosphoglycerate kinase (3PGK) (12) (12) – glyceraldehyde 3-phosphate (12) dehydrogenase (GAPDH) Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP → 2 glyceraldehyde 3- + + phosphate (GA3P) + 4 H + 12 NADP + 18 ADP + 16 Pi *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo)

Photosynthesis The Three Stages of the Calvin Cycle of Reverse of glycolysis: GA3P converted to DHAP by triose phosphate (TPI); 10 enzymes CO2 Assimilation GA3P and DHAP converted to (6) fructose 1,6-bisphosphate (Fru 1,6-P2) via aldolase, the to Fru (6) 6-P via fructose 1,6- bisphosphatase, then Glc 6-P (6) via phosphoglucoisomerase (PGI). Rubisco (10) (6) (2) 10/12 glyceraldehyde-3-phosphate products (30 carbons) MUST be used to regenerate 6 ribulose-1,5- bisphosphate (30 carbons). TPI DHAP ⇌ (12) (12) Aldolase

Fructose-1,6- Fru 1,6P2

bisphosphatase ⇌ Pi (12) chloroplast isoforms Fru6P (12) – 3-phosphoglycerate kinase (3PGK) PGI ⇌ (12) (12) – glyceraldehyde 3-phosphate Glc6P (12) dehydrogenase (GAPDH) • 12 glyceraldehyde-3-phosphate are produced during the Calvin cycle in the assimilation of 6 carbons into Glc. • 2/12 glyceraldehyde-3-phosphate products are pulled out of the cycle and used for Glc6P, then to other carbos.

5 Photosynthesis Third Stage of Calvin Cycle Stage 3: Regeneration of Ru 1,5-P2 1. We have 3-carbon compounds and need 5-carbon compounds: put 2 together to make 6 (stop at Fru6P), then can pull 2 carbons from 6 (leaving 4) and with another 3 carbon compound make a 5-carbon sugar. DO THIS TWICE. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P). From 4 GA3P

10àuse 6 GA3P 4 GA3P à 2 Fru 6-P 2 GA3P à 2 Xyl 5-P

transketolase

2 2 2 2

Photosynthesis Third Stage of Calvin Cycle Stage 3: Regeneration of Ru 1,5-P2 1. We have 3-carbon compounds and need 5-carbon compounds: put 2 together to make 6 (stop at Fru6P), then can pull 2 carbons from 6 (leaving 4) and with another 3 carbon compound make a 5-carbon sugar. DO THIS TWICE. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P). From 4 GA3P

10àuse 6 GA3P 4 GA3P à 2 Fru 6-P 2 GA3P à 2 Xyl 5-P

transketolase

2 2 2 2

6 Photosynthesis Transketolase: exchange of 2C from ketose to an aldose acceptor

Uses Pyrophosphate (TPP) as the

•Contains thiazolium anion for nucleophilic attack on carbonyls of ketose Aldose e.g., erythrose •Also used by: e.g., fructose 6-phosphate 4-phosphate – in acetyl CoA formation – pyruvate decarboxylase in

– a-ketoglutarate e.g., glyceraldehyde 3-phosphate dehydrogenase in CAC – transketolase in pentose phosphate pathway e.g., xyulose 5-phosphate

Stage 3 4à use 2 GA3P Photosynthesis 2à0 GA3P left

aldolase sedoheptulose 1,7- transketolase bisphosphatase

⇌ ⇌

2 2 2 2 2 2 2 2. We have two 4-carbon Ery 4-P and still have 4/12 GA3Ps. Do it again; aldol condensation with DHAP to get Sedoheptulose 1,7-bisphosphate (Sed 1,7-P2).

3. If Sed 1,7-P2 loses the C7-phosphate, as a ketose, we can pull 2 carbons off and put on our remaining 2 ketoses (GA3P) 6 Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7- phosphate (S7P) via sedoheptulose 1,7-bisphosphatase 2 from – This enzyme is unique to plants. Fru6-P 2 from Finally, RuP phosphorylated to ribulose Ery4-P 1,5-bisphosphate by 2+2=4 6 6 – This enzyme is also unique to plants and regulated by NADPH

7 Stage 3 4à use 2 GA3P Photosynthesis 2à0 GA3P left

aldolase sedoheptulose 1,7- transketolase bisphosphatase

⇌ ⇌

2 2 2 2 2 2 2 2. We have two 4-carbon Ery 4-P and still have 4/12 GA3Ps. Do it again; aldol condensation with DHAP to get Sedoheptulose 1,7-bisphosphate (Sed 1,7-P2).

3. If Sed 1,7-P2 loses the C7-phosphate, as a ketose, we can pull 2 carbons off and put on 2 our remaining 2 ketoses (GA3P) 6 Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7- phosphate (S7P) via sedoheptulose 1,7-bisphosphatase 2 from – This enzyme is unique to plants. Fru6-P 2 from Finally, RuP phosphorylated to ribulose Ery4-P 1,5-bisphosphate by phosphoribulokinase 2+2= 4 6 6 – This enzyme is also unique to plants and regulated by NADPH

Photosynthesis 2 2 Stage 3: Summary Regeneration Steps for 2 10/12 of Glyceraldehyde-3- phosphate Products of the Calvin Cycle (Overview) ribulose-5- 2 2 phosphate epimerase 6 6

2 Stage 3: Addition of Phosphate from ATP to ribulose-5- phosphate Ribulose-5-Phosphate epimerase Commits Product to Calvin Cycle 2 2

8 Photosynthesis

Stoichiometry and Energetics of CO2 Assimilation in the Calvin Cycle & NET Reaction for Photosynthesis: + 6 CO2 + 12 NADPH + 12 H + Stage 1/2 12 H2O + 18 ATP → + + 1 C6H12O6 + 2 H + 12 NADP + + 18 ADP + 18 Pi + 18 H

This is 3:2 ATP:NADPH! (from 8 photons) For every NADPH, 4 photons needed 12 Pi ∴ need 12x4=48 photons to make 1 Glc

Stage 1/2 2 Pi 6Ru5Pà12GA3P Stage 2 DG°’ = –56 kcal/mol (end) Stage 2 (end) 2GA3Pà1Glc DG°’ = –10 kcal/mol Stage 3 Stage 3

10GA3Pà 6Ru5P 1 Pi DG°’ = –38 kJ/mol Glucose NET: –104/6 = –17.3 per CO2

Photosynthesis

Photosynthesis: Control by Light and CO2 to Glucose • The assembly of one of glucose requires the capture of roughly 48 photons. – H+ move from the stroma to the thylakoid – creates alkaline conditions in the stroma How H+ and Mg2+ • Accompanied by Mg2+ transport from Movement Assist thylakoid to stroma in Activation of • Enzyme for photosynthesis and CO2 Photosynthesis assimilation more active in the alkaline & and CO2 high [Mg2+] conditions of the stroma Assimilation • This is a source of coordinated regulation by several enzymes: – Target enzymes regulated by NADPH, Mg2+, and Glucose 100 pH, or all three are: ase • Rubisco 2

• fructose 1,6-bisphosphatase 60 • seduloheptose 1,7-bisphosphatase ase, Sed1,7P ase, • glyceraldehyde 3-phosphate dehydrogenase 2

• ribulose 5-phosphate kinase Activity Relative 30 (phosphoribulokinase) How are these regulated by NADPH? Fru1,6P Rubisco,

9 Photosynthesis

Photosynthesis: Control from Light and CO2 to Glucose Target enzymes regulated by Fd:NADPH: fructose 1,6-bisphosphatase seduloheptose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenase ribulose 5-phosphate kinase (phosphoribulokinase) Rubisco activase If oxidized (Cys residues in Cys-Cys form) à enzymes are inactive. In light, photosystem I sends e– to ferredoxin, which sends them to , which donates them to disulfide bonds to reduce them to free Cys.

Excess reduced Fd is Light Activation of FOUR Calvin Cycle Enzymes via only available after Electron-Driven Reduction of Cys-Cys Crosslinks its use for NADPH production

fructose 1,6-bisphosphatase sedoheptulose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenase ribulose 5-phosphate kinase

Photosynthesis

Assimilation of CO2 into Biomass

• Plant cells: use 3-C intermediates for further synthesis – Glyceraldehyde 3-phosphate (G3P) is the most important one.

– made from CO2, H2O, plus ATP and NADPH from photosynthesis

10