Rubisco

Question 1

Rubisco catalyses both carboxylation and oxygenation reactions

Answer 1

• Ribulose 1,5-bisphosphate carboxylase /oxygenase (Rubisco) catalyses the reaction between Ribulose 1,5-bisphosphate (RuBP) and CO2 or O
  ^ Therefore it functions as either a carboxylase or an oxygenase

-Carboxylation produces two molecules of 3-phosphoglycerate (3-PGA)Oxygenation produces one 3-PGA and one 2-phosphogylcolate (2-PG)
^ Recycling the wasteful oxygenation product 2-PGcosts energy and releases fixed CO

Question 2

Rubisco does not discriminate well between O2 and CO2

Answer 2

It wants to add CO2 but sometimes adds O2 instead - struggles to discriminate

It evolved when the atmosphere was mostly CO2 and it did not need to discriminate therefore now in a high O2 atmosphere it is less efficient

Many other carboxylases including PEPC bind to and use bicarbonate(HCO3-) as a substrate, thus avoiding competition by O2

Plants are approx 100x more specific for CO2 than O2
^ so for every 100 reactions 99 would be CO2 and 1 O2 if they were in the same abundance in the atmosphere.
BUT
There is about 25x more O2 than CO2 in the atmosphere
^ So in fact 1 in 4 reactions use O2

Question 3

Rubisco is found in a variety of forms

Answer 3

located on the stromal side of the thylakoid membrane

why are there only small subunits in form 1?
Reactions happen on the large subunit – it appears that the small subunits evolved later to regulate the large subunit. This is likely related to the fact that many bacteria and archaea are able to switch between being autotrophic and heterotrophic behaviours so it is likely that the small subunits evolved later for optimisation in autotrophic specialists.

• Form I: Plants and most cyanobacteria have a hexadecameric form consisting of 8 Large subunits and 8 Small subunits (L8S8)
  ^ Only Form I has small subunits

-Form II: Some bacteria and some dinoflagellate algae have form II, made of dimers of L, ranging fromL2 to L8

-Form III: Some archaea. Form III is found as L2,(L2)4 or (L2)5. Form III also serves a non-photosynthetic function

-Form IV: Rubisco-Like Proteins with no catalytic activity. Some bacteria. Found as L2

The large rubisco subunit is extremely well conserved across so is often used to compare phylogenetic relationships between organisms in the form of genetic barcoding

Question 4

Different forms of Rubisco have different catalytic properties

Answer 4

(see graphs)

making Rubisco more specific reduces efficiency so there is a trade off between speed and specificity.

• Specificity factor indicates the specificity of the enzyme for CO2 relative to O2. Higher means more specific for CO2.
• Rubisco in red algae is more specific but slower than that in plant

-In many organisms, the enzyme cannot be both fast and specific

• Rubisco in cyanobacteria algae is faster but less specific than that in plants

Question 5

Rubisco’s catalytic inefficiency means that plants invest hugely in Rubisco

Answer 5

• RUBISCO fixes 1015 g C per year, about 1/7th of all atmospheric CO2
• RUBISO is slow, catalysing 3 – 10 reactions per second (compare to carbonic anhydrase, at 500,000 rxns / sec)
• To make up for Rubisco’s catalytic deficiencies, plants make LOTS of it. In C3 plants, 50% of soluble leaf protein can be Rubisco
• Algae and C4 plants have carbon-concentrating mechanisms so can get by with much less Rubisco protein.

Question 6

Rubisco activity is regulated at many levels including by light and metabolites

Answer 6

Transcription:
- Transcription of small subunit genes in nuclear genome
- Transcription of large subunit gene in plastid genome

Assembly
- The 16-subunit holoenzyme is assembled sequentially and involves the contributions of several dedicated chaperone proteins

Reversible inhibition
- Several inhibitory sugar phosphates bind to Rubisco.
- Inhibitor removal and enzyme activation tunes Rubisco activity to environmental conditions

Question 7

In plants Rubisco assembles from large and small subunits in the chloroplast

Answer 7

(see diagram)

1. LSU is encoded by a single gene in the multicopy chloroplast genome
2. SSU is encoded by several genes in the nuclear genome
3. Chaperones including DNAJ, BSDII and RbcX are needed for holoenzyme assembly
4. Rubisco activase is needed to activate Rubisco

Question 8

A lysine in the active site must be carbamylated for activity

Answer 8

(see notes for equation and diagram)

Question 9

Rubisco is subject to inactivation which can be reversed by Rubisco activase

Answer 9

Rubisco activase (RA or RCA) uses the energy of ATP to remove inhibitors, thereby activating Rubisco

Question 10

Rubisco activase is heat labile. Heat-stabilized RA can confer thermotolerance

Answer 10

In vivo and in vitro, Rubisco is limited at higher temperatures by Rubisco activase’s heat sensitivity

Seed development is limited at 26°C in wild type plants, but restored in plants with heat-stabilized Rubisco activase

Modification of plants to increase thermo-stability of rubisco activase allows plants to grow faster at higher temperatures