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tightly binds ribulose bisphosphate (RuBP) at the active site as a “dead end” complex , with the closed. conformation, and is inactive in catalysis[r]

Photosynthesis: Calvin Cycle

Copyright © 1999-2008 by Joyce J Diwan All rights reserved

Light reactions: Energy of light is conserved as

 “high energy” phosphoanhydride bonds of ATP

 reducing power of NADPH

Proteins & pigments responsible for the light reactions

are in thylakoid (grana disc) membranes

Light reaction pathways will be not be presented here grana disks

(thylakoids)

stroma compartment

2 outer

membranes

Chloroplast Photosynthesis

takes place in chloroplasts It includes light reactions and

reactions that are not directly

The free energy of cleavage of ~P bonds of ATP, and reducing power of NADPH, are used to fix and reduce

CO2 to form carbohydrate

Enzymes & intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat

analogous to the mitochondrial matrix grana disks

(thylakoids)

stroma compartment

2 outer

membranes

Chloroplast Calvin Cycle,

earlier designated the photosynthetic "dark reactions," is now called the

carbon reactions

Ribulose Bisphosphate Carboxylase (RuBP Carboxylase), catalyzes CO2 fixation:

ribulose-1,5-bisphosphate + CO2  3-phosphoglycerate

Because it can alternatively catalyze an oxygenase reaction, the enzyme is also called RuBP Carboxylase/Oxygenase

(RuBisCO) It is the most abundant enzyme on earth

Ribulose-1,5-bisphosphate (RuBP)

OH H2C

C H

C C

OH H

H2C OPO3

2-OPO3 2-O

3-Phosphoglycerate (3PG)

OH H2C

C H

C O O

OPO 3

-RuBP Carboxylase - postulated mechanism:

Extraction of H+ from C3 of ribulose-1,5-bisphosphate promotes formation of an enediolate intermediate

Nucleophilic attack on CO2 leads to formation of a 

-keto acid intermediate, that reacts with water and cleaves to form molecules of 3-phosphoglycerate

O H H2C

C H C C O H H

H2C O P O 32

O P O 32 O

O H H2C

C H

C C

O H

H2C O P O32

O P O32

 O

H+ O H

H 2C C H

C C

O

H 2C O P O32

O P O32 H O C O2 C O2

O H H 2C

C H

C

O P O 32 O

O

H2O

1

5

Transition state analogs of the postulated -keto acid

intermediate bind tightly to RuBP Carboxylase and inhibit

its activity

Examples: 2-carboxyarabinitol-1,5-bisphosphate (CABP, above right) & carboxyarabinitol-1-phosphate (CA1P)

2-Carboxyarabinitol-1,5-bisphosphate (inhibitor)

OH H2C

C H C C OH H

H2C OPO32

OPO32 HO CO2

Proposed -keto acid intermediate

OH H2C

C H

C C

O

H2C OPO 32

 8 large catalytic subunits (L, 477 residues, blue, cyan)  8 small subunits (S, 123 residues, shown in red).

Some bacteria contain only the large subunit, with the smallest functional unit being a homodimer, L2

Roles of the small subunits have not been clearly defined There is some evidence that interactions between large & small subunits may regulate catalysis

RuBisCO PDB 1RCX

RuBisCO PDB 1RCX

RuBP

Carboxylase

in plants is a complex

Large subunits within

RuBisCO are arranged as

antiparallel dimers, with the N-terminal domain of one

monomer adjacent to the C-terminal domain of the other Each active site is at an

interface between monomers within a dimer, explaining the minimal requirement for a

dimeric structure

The substrate binding site is at the mouth of an -barrel

domain of the large subunit

Most active site residues are polar, including some charged amino acids (e.g., Thr, Asn, Glu, Lys)

ribulose-1,5-bisphosphate

PDB 1RCX

"Active" RuBP Carboxylase has a carbamate that binds an essential Mg++ at the active site

The carbamate forms by reaction of HCO3 with the  -amino group of a lysine residue, in the presence of Mg++

HCO3 that reacts to form carbamate is distinct from CO

2 that binds to RuBP Carboxylase as substrate

Mg++ bridges between oxygen atoms of the carbamate &

substrate CO2

Carbamate Formation

with RuBP Carboxylase Activation

Enz-Lys NH3+ HN C

O

O

+ HCO3 + H

2O + H+

Binding of either RuBP or a transition state analog to RuBP Carboxylase causes a conformational change to a "closed" conformation in which access of solvent

water to the active site is blocked

RuBP Carboxylase (RuBisCO) can spontaneously

deactivate by decarbamylation

In the absence of the carbamate group, RuBisCO

tightly binds ribulose bisphosphate (RuBP) at the active site as a “dead end” complex, with the closed

conformation, and is inactive in catalysis

RuBP Carboxylase Activase is an ATP hydrolyzing

(ATPase) enzyme that causes a conformational change in RuBP Carboxylase from a closed to an open state

This allows release of tightly bound RuBP or other sugar phosphate from the active site, and carbamate formation Since photosynthetic light reactions produce ATP, the

ATP dependence of RuBisCO activation provides a

mechanism for light-dependent activation of the enzyme The activase is a member of the AAA family of ATPases, many of which have chaperone-like roles

RuBP Carboxylase Activase is a large multimeric protein

When O2 reacts with ribulose-1,5-bisphosphate, the

products are 3-phosphoglycerate plus the 2-C compound 2-phosphoglycolate

This reaction is the basis for the name RuBP Carboxylase/Oxygenase (RuBisCO)

OH H 2C

C H

C O

O

OPO 32



H 2C C

OPO 32

O  O

3 -p h o s p h o - p h o s p h o g ly c o la te g ly c e te

Photorespiration:

O2 can compete with CO2

The complex pathway that partly salvages C from 2-phosphoglycolate, via conversion to 3-phosphoglycerate,

involves enzymes of chloroplasts, peroxisomes & mitochondria

This pathway recovers 3/4 of the C as 3-phosphoglycerate The rest is released as CO2

Photorespiration is a wasteful process, substantially

reducing efficiency of CO2 fixation, even at normal ambient CO2

OH H 2C

C H

C O O

OPO 32



H 2C C

OPO 32

O 

O

3 -p h o s p h o - p h o s p h o g ly c o la te g ly c e r a te

Photorespiration:

 Most plants, designated C3, fix CO2 initially via RuBP Carboxylase, yielding the 3-C 3-phosphoglycerate

 Plants designated C4 have one cell type in which phosphoenolpyruvate (PEP) is carboxylated via the

enzyme PEP Carboxylase, to yield the 4-C oxaloacetate Oxaloacetate is converted to other 4-C intermediates that

are transported to cells active in photosynthesis, where

C4 plants maintain a high ratio of CO2/O2 within

photosynthetic cells, thus minimizing photorespiration Research has been aimed at increasing expression of

Continuing with Calvin Cycle:

The normal RuBP Carboxylase product, 3-phospho-glycerate is converted to glyceraldehyde-3-P

Phosphoglycerate Kinase catalyzes transfer of Pi from

ATP to the carboxyl of 3-phosphoglycerate (RuBP

Carboxylase product) to yield 1,3-bisphosphoglycerate

OH H 2 C

C H

C O

O

OPO 3 



OH H 2 C

C H

C O PO

2

O

OPO 3 

OH H 2 C

C H

CHO

OPO 3 

A T P A D P N A D P H N A D P +

P i

1 , - b i s p h o s p h o - g l y c e r a t e - p h o s p h o -

g l y c e r a t e g l y c e r a l d e h y d e - - p h o s p h a t e

P h o s p h o g l y c e r a t e K i n a s e

Glyceraldehyde-3-P Dehydrogenase catalyzes reduction of the carboxyl of 1,3-bisphosphoglycerate to an aldehyde, with release of Pi, yielding glyceraldehyde-3-P

This is like the Glycolysis enzyme running backward, but the chloroplast Glyceraldehyde-3-P Dehydrogenase uses

NADPH as e donor, while the cytosolic Glycolysis

enzyme uses NAD+ as e acceptor

OH H 2 C

C H

C O

O

OPO 3 



OH H 2C

C H

C O PO

2 

O

OPO 32 

OH H 2 C

C H

CHO

OPO 3 

A T P A D P N A D P H N A D P +

P i

1 , - b i s p h o s p h o - g l y c e r a t e - p h o s p h o -

g l y c e r a t e g l y c e r a l d e h y d e - - p h o s p h a t e

P h o s p h o g l y c e r a t e K i n a s e

Continuing with Calvin Cycle:

A portion of the glyceraldehyde-3-P is converted back to

ribulose-1,5-bisP, the substrate for RuBisCO, via

reactions catalyzed by:

Triose Phosphate Isomerase, Aldolase, Fructose Bisphosphatase, Sedoheptulose Bisphosphatase, Transketolase, Epimerase, Ribose Phosphate

Isomerase, & Phosphoribulokinase

Many of these are similar to enzymes of Glycolysis,

Summary of Calvin cycle:

3 5-C ribulose-1,5-bisP (total of 15 C) are carboxylated (3 C added), cleaved, phosphorylated, reduced, &

dephosphorylated, yielding

6 3-C glyceraldehyde-3-P (total of 18 C) Of these: 1 3-C glyceraldehyde-3-P exits as product

5 3-C glyceraldehyde-3-P (15 C) are recycled back into 3 5-C ribulose-1,5-bisphosphate

C3 + C3  C6

C3 + C6  C4 + C5

C3 + C4  C7

C3 + C7  C5 + C5

Overall:

C3  3 C5

Enzymes:

TI, Triosephosphate Isomerase

AL, Aldolase

FB, bisphosphatase

SB, Bisphosphatase

TK, Transketolase

EP, Epimerase

IS, Isomerase

PK,

ribulokinase

TK

EP

PK

glyceraldehyde-3-P dihydroxyacetone-P

fructose-6-P

xyulose-5-P + erythrose-4-P

sedoheptulose-7-P

3 CO2 + ATP + NADPH 

glyceraldehyde-3-P + ADP + Pi + NADP+

Glyceraldehyde-3-P may be converted to other CHO:

• metabolites (e.g., fructose-6-P, glucose-1-P) • energy stores (e.g., sucrose, starch)

• cell wall constituents (e.g., cellulose)

Glyceraldehyde-3-P can also be utilized by plant cells as carbon source for synthesis of other compounds such as fatty acids & amino acids

g l y c e r a l d e h y d e - - p h o s p h a t e

OH H 2 C

C H

CHO

OPO 3 

O C

O

c a r b o n d i o x i d e

There is evidence for multienzyme complexes of Calvin Cycle enzymes within the chloroplast stroma

Positioning of many Calvin Cycle enzymes close to the enzymes that produce their substrates or utilize their

reaction products may increase efficiency of the pathway grana disks

(thylakoids)

stroma compartment

2 outer

membranes

Regulation of Calvin Cycle

Regulation prevents the Calvin Cycle from being

active in the dark, when it might function in a

futile cycle with Glycolysis & Pentose Phosphate Pathway, wasting ATP & NADPH

Light-activated e transfer is linked to pumping of H+

into thylakoid disks pH in the stroma increases to about Alkaline pH activates stromal Calvin Cycle enzymes

RuBP Carboxylase, Fructose-1,6-Bisphosphatase & Sedoheptulose Bisphosphatase

The light-activated H+ shift is countered by Mg++ release

from thylakoids to stroma RuBP Carboxylase (in stroma) requires Mg++ binding to carbamate at the active site

stroma

(alkaline)

Chloroplast

H2O  OH



+ H+

h

(acid inside thylakoid disks)

Some plants synthesize a transition-state inhibitor, carboxyarabinitol-1-phosphate (CA1P), in the dark

disulfide

Thioredoxin f PDB 1FAA

Thioredoxin is a small protein with a disulfide that is reduced in chloroplasts via light-activated

During illumination, the thioredoxin disulfide is reduced to

a dithiol by ferredoxin, a constituent of the photosynthetic

light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase

Reduced thioredoxin activates several Calvin Cycle

enzymes, including Fructose-1,6-bisphosphatase,

Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase

Activase, by reducing disulfides in those enzymes to thiols

th

io

re

do

xi

n

 S  S

th

io

re

do

xi

n

 SH  SH

|

ferredoxinRed ferredoxinOx

Ferredoxin- Thioredoxin

Diagram