RuBisCo.ppt
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Rubisco, or ribulose-1,5-bisphosphate carboxylase/oxygenase, is a crucial enzyme in the Calvin cycle responsible for catalyzing the attachment of CO2 to ribulose-1,5-bisphosphate, resulting in the formation of 3-phosphoglyceric acid. The enzyme is composed of large subunits encoded by the chloroplast genome and small subunits encoded by the nuclear genome, requiring precise assembly and regulation for activity. Rubisco activity is influenced by environmental factors such as light and CO2 concentration, with regulatory mechanisms involving activation by rubisco activase and inhibition through naturally occurring regulators.
![Rubisco activity and CO2 concentration
• If [CO2] > 50ppm, carboxylase activity
• If [CO2] < 50ppm, oxygenase activity](https://image.slidesharecdn.com/rubisco-221102182042-2fdb8e23/85/RuBisCo-ppt-5-320.jpg)
RuBisCo.ppt
- 1. RuBisCo Synthesis, Assembly, Mechanism, and Regulation
- 2. Ribulose 1,5-Bisphosphate Carboxylase (Rubisco) Reactions: RuBP (5C) CO2 + H2O Rubisco 2 X 3-P-Glycerate (3C) + 2 H+ RuBP (5C) Rubisco O2 + H2O 1 X 3-P-Glycerate (3C) + 2H+ + 1 X 2-P-Glycolate (2C)
- 3. The Calvin-Benson Cycle Enzyme rubisco attaches CO2 to RuBP – Forms two 3-carbon 3-phosphoglyceric acid molecules – 3-phosphoglyceric acid molecules combines with ATP and NADPH to form Glyceraldehyde 3-phosphate • Glyceraldehyde 3-phosphate receive a phosphate group from ATP, and hydrogen and electrons from NADPH – Two Glyceraldehyde 3-phosphate combine to form a Ribulose-5- Phosphate – Ribulose-5-Phosphate, with an additional ATP molecule converted to Ribulose-1,5-Bisphosphate – Ribulose-1,5-Bisphosphate with the help of RuBisCo and CO2 produces two 3-carbon 3-phosphoglyceric acid molecules
- 5. Rubisco activity and CO2 concentration • If [CO2] > 50ppm, carboxylase activity • If [CO2] < 50ppm, oxygenase activity
- 7. Located in chloroplasts: 6 Large Subunits (LSU), 55 kDa. rbcL gene Encoded on chloroplast genome Contains substrate (CO2 and O2) binding site. 6 Small Subunits (SSU), 12-18 kDa. RbcS gene Encoded in nuclear genome as gene family Synthesized as precursor, 20 Kda, with plastid transit sequence Transported to chloroplasts Possibly for regulation and assembly Structure of the RuBisCO
- 8. RUBISCO • The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase • Rubisco is found in most autotrophic organisms from prokaryotes (photosynthetic and chemoautotrophic bacteria, cyanobacteria and archaea) to eukaryotes (various algae and higher plants). • Metabolically Important enzyme in Calvin cycle • RuBisCO, is the most abundant globular protein in the world • rubisco makes up 20-25% of the soluble protein in leaves
- 9. • RuBisCo has a molecular weight of 490,000 Daltons • Eight large subunits (53000Da) and eight small subunits(12000Da) 6 large subunits (blue/ violet) highly conserved, occurring as dimers Codes on chloroplast DNA; one catalytic site per dimer
- 10. • 6 small subunits, • (gold); required for function of large subunits • Coded in nucleus as multigene family; • The SSU are synthesized as precursors in the cytoplasm , processed and transported to the organelle where they bind to LSU • different genes expressed in response to environmental changes, including light. • Also regulated by biological clock, giving it a light: dark periodicity in abundance and activity.
- 11. • The catalytic site is located at the face of an alpha/beta barrel domain in the carboxyl terminus of the larger subunit
- 12. RUBISCO gene organization and expression • rbcS – Located in the nucleus – Contains one to three introns – encoding 120 amino acids – Expression is controlled by light – Transcription of rbcS occur in the photosynthetic tissues – mRNA are imported in to chloroplast (ATP dependent)
- 13. • rbcL – Present in chloroplast genome – Do not contain introns – Codes 475 amino acids – transcribed by the plastid-encoded plastid RNA polymerase (PEP) – The translation of rbcL mRNA is enhanced by light • syntheses of SSU and LSU are optimally regulated via intracellular crosstalk between the nucleus and the chloroplast
- 15. Coordinate expression of nuclear and chloroplast genes
- 16. Accumulation of Rubisco requires stoichiometric assembly of subunits
- 17. Folding and assembly into theRuBisCO holoenzyme in chloroplasts • Import into chloroplasts and processing • Mature S-subunits are assembled with plastid- synthesized L-subunits into the Rubisco holoenzyme • L-subunits are also stably associated with a large oligomeric protein-chaperonin 60 (cpn6O) RuBisCO binding protein • Rubisco L-subunits are specifically associated with cpn60 before assembly into holoenzyme
- 18. • The assembly of Rubisco is accelerated by ATP • Requires Mg2+, cpn60 and putative intermediates in the assembly of the Rubisco holoenzyme. • LSU is assembled with SSU after dissociation from the chaperone complex to construct a holoenzyme
- 19. CHAPERONIN-MEDIATED FOLDING AND ASSEMBLY OF RUBISCO • Requires the cpn 10 co chaperonin • Two generic folding environments – Permissive • unassisted spontaneous folding can occur • Require only cpn60 – Non permissive • spontaneous folding cannot occur • Requires both cpn60 and cpn10
- 20. THE RUBISCO ACTIVE SITE • active site – C-terminal barrel domain of one L-subunit of the dimer and the N-terminal domain of the second L- subunit of the dimer. • Inactive enzyme – Site for cofactors and bisphosphate substrate – After binding of all regulatory elements Closure of the loops brings together aminoacids that are critical for catalysis
- 21. Regulation of Activity and Role of Rubisco Activase • Activation of the enzyme involving carbamylation of an active site Lys (Lys-201 in spinach Rubisco) residue by CO2 • Carbamino group in close proximity to two adjacent acidic residues, Asp- 203 and Glu- 204, provides a site for the essential Mg2+ ion to bind
- 22. • catalytic mechanism of the carboxylation of ribulosebisphosphate – formation of an enediol intermediate of the bisphosphate substrate – C2,C3-enediol reacts with CO2 at the C2 position, forming a six-carbon intermediate – hydrolytically cleaved to two molecules of 3- phosphoglycerate (3P-glycerate).
- 23. Activation of RUBISCO • Rubisco cycles between active and inactive form. • Active form requires a bound Mg2+ ion, light and high pH. • Substrate CO2 molecule participates in Mg2+ binding to active site. • CO2 molecule binds reversibly to lysine residue forming carbamate adduct • Activation facilitated by the enzyme rubisco activase. • In the dark, carbamate adduct disassociates from active site. R 1,5-BP then binds tightly to active site and inhibits enzyme
- 25. REGULATION OF RUBISCO ACTlVlTY • 2'-carboxy arabinitol 1-phosphate (2CAlP) is a naturally occurring inhibitor • It accumulates in the dark and in low-light conditions, binding to the activated form of the enzyme • Substrate inhibiton – xylulosebisphosphate, which differs from ribulose- bisphosphate – Irreversable inhibition • enzyme must reactivate with CO2 and Mg2+ before catalysis can proceed
Editor's Notes
- #16: L and S subunits of Rubisco are prototypical examples of coordinated gene expression