RuBisCo.ppt

Dr.S.KARTHIKUMAR
Associate Professor
Department of Biotechnology
Kamaraj College of Engineering and Technology, K.Vellakulam-625701, TN, India
Email: [email protected]

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 rubiscoattaches CO
2to 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 CO
2
produces two 3-carbon 3-phosphoglyceric acid molecules

4. RUBISCO Reaction Mechanisms
carboxylase
oxygenase

5. Rubisco activity and CO
2concentration
•If [CO
2] > 50ppm, carboxylase activity
•If [CO
2] < 50ppm, oxygenase activity

7. Located in chloroplasts:
6 Large Subunits (LSU), 55 kDa.rbcL gene
Encoded on chloroplast genome
Contains substrate (CO2and 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
•Rubiscois 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
•rubiscomakes 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 rbcLmRNA 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
theRuBisCOholoenzymein
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
oligomericprotein-chaperonin60 (cpn6O)
RuBisCObinding protein
•RubiscoL-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
RubiscoActivase
•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 Mg
2+
ion, light
and high pH.
•Substrate CO
2molecule participates in Mg
2+
binding to active site.
•CO
2molecule 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 arabinitol1-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
–Irreversableinhibition
•enzyme must reactivate with CO2 and Mg2+ before
catalysis can proceed