Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO,
is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric
carbon dioxide is converted by plants to energy-rich molecules such as glucose. In chemical terms,
it catalyzes the carboxylation of ribulose-1,5-bisphosphate. It is probably the most abundant protein on
Earth.
RuBisCO vs alternative carbon fixation pathways RuBisCO is important biologically because
it catalyzes the primary chemical reaction by which inorganic carbon enters the biosphere.
While many autotrophic bacteria and archaea fix carbon via the reductive acetyl CoA pathway,
the 3-hydroxypropionate cycle, or the reverse Krebs cycle, these pathways are relatively
smaller contributors to global carbon fixation than that catalyzed by RuBisCO. Phosphoenolpyruvate
carboxylase, unlike RuBisCO, only temporarily fixes carbon. Reflecting its importance, RuBisCO
is the most abundant protein in leaves, accounting for 50% of soluble leaf protein in C3 plants
and 30% of soluble leaf protein in C4 plants. Given its important role in the biosphere,
the genetic engineering of RuBisCO in crops is of continuing interest.
Structure
In plants, algae, cyanobacteria, and phototrophic and chemoautotrophic proteobacteria, the enzyme
usually consists of two types of protein subunit, called the large chain and the small chain.
The large-chain gene is part of the chloroplast DNA molecule in plants. There are typically
several related small-chain genes in the nucleus of plant cells, and the small chains are imported
to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast
membrane. The enzymatically active substrate binding sites are located in the large chains
that form dimers as shown in Figure 1 in which amino acids from each large chain contribute
to the binding sites. A total of eight large-chains and eight small chains assemble into a larger
complex of about 540,000 Da. In some proteobacteria and dinoflagellates, enzymes consisting of
only large subunits have been found. Magnesium ions are needed for enzymatic activity.
Correct positioning of Mg2+ in the active site of the enzyme involves addition of an
"activating" carbon dioxide molecule (CO 2) to a lysine in the active site. Formation
of the carbamate is favored by an alkaline pH. The pH and the concentration of magnesium
ions in the fluid compartment increases in the light. The role of changing pH and magnesium
ion levels in the regulation of RuBisCO enzyme activity is discussed below.
Enzymatic activity
As shown in Figure 2, RuBisCO is one of many enzymes in the Calvin cycle.
Substrates During carbon fixation, the substrate molecules
for RuBisCO are ribulose-1,5-bisphosphate, carbon dioxide. RuBisCO also catalyses a reaction
between ribulose-1,5-bisphosphate and molecular oxygen (O
2) instead of carbon dioxide (CO 2).
Products When carbon dioxide is the substrate, the
product of the carboxylase reaction is a highly unstable six-carbon phosphorylated intermediate
known as 3-keto-2-carboxyarabinitol-1,5-bisphosphate, which decays virtually instantaneously into
two molecules of glycerate-3-phosphate. The extremely unstable molecule created by the
initial carboxylation was unknown until 1988, when it was isolated. The 3-phosphoglycerate
can be used to produce larger molecules such as glucose. When molecular oxygen is the substrate,
the products of the oxygenase reaction are phosphoglycolate and 3-phosphoglycerate. Phosphoglycolate
is recycled through a sequence of reactions called photorespiration, which involves enzymes
and cytochromes located in the mitochondria and peroxisomes. In this process, two molecules
of phosphoglycolate are converted to one molecule of carbon dioxide and one molecule of 3-phosphoglycerate,
which can reenter the Calvin cycle. Some of the phosphoglycolate entering this pathway
can be retained by plants to produce other molecules such as glycine. At ambient levels
of carbon dioxide and oxygen, the ratio of the reactions is about 4 to 1, which results
in a net carbon dioxide fixation of only 3.5. Thus, the inability of the enzyme to prevent
the reaction with oxygen greatly reduces the photosynthetic capacity of many plants. Some
plants, many algae, and photosynthetic bacteria have overcome this limitation by devising
means to increase the concentration of carbon dioxide around the enzyme, including C4 carbon
fixation, crassulacean acid metabolism, and the use of pyrenoid.
Rate of enzymatic activity Some enzymes can carry out thousands of chemical
reactions each second. However, RuBisCO is slow, being able to fix only 3-10 carbon dioxide
molecules each second per molecule of enzyme. The reaction catalyzed by RuBisCO is, thus,
the primary rate-limiting factor of the Calvin cycle during the day. Nevertheless, under
most conditions, and when light is not otherwise limiting photosynthesis, the speed of RuBisCO
responds positively to increasing carbon dioxide concentration. However, our descriptive knowledge
will become more usable when we can translate them into quantitative models that can enable
us to calculate the outcome of the reaction under a given condition. Since RubisCO reacts
with RuBP first to produces enediol and next with CO
2 that after some intermediate changes produces PGA, a biochemical model is developed to represent
the effects of these steps quantitatively. Since carboxylation or fixation of CO
2 is possible only after the synthesis of enediol, thus it is suggested that the role
of RubisCO is to produce enediol that is carboxylase and oxygenase. Accordingly, RubisCO is called
enolase-phosphglycerase since it is neither carboxylase nor oxygenase.
Regulation of its enzymatic activity RuBisCO is usually only active during the
day as ribulose 1,5-bisphosphate is not regenerated in the dark. This is due to the regulation
of several other enzymes in the Calvin cycle. In addition, the activity of RuBisCO is coordinated
with that of the other enzymes of the Calvin cycle in several ways.
Regulation by ions Upon illumination of the chloroplasts, the
pH of the stroma rises from 7.0 to 8.0 because of the proton gradient created across the
thylakoid membrane. At the same time, magnesium ions move out of the thylakoids, increasing
the concentration of magnesium in the stroma of the chloroplasts. RuBisCO has a high optimal
pH and, thus, becomes "activated" by the addition of carbon dioxide and magnesium to the active
sites as described above. Regulation by RuBisCO activase
In plants and some algae, another enzyme, RuBisCO activase, is required to allow the
rapid formation of the critical carbamate in the active site of RuBisCO. RuBisCO activase
is required because the ribulose 1,5-bisphosphate substrate binds more strongly to the active
sites lacking the carbamate and markedly slows down the "activation" process. In the light,
RuBisCO activase promotes the release of the inhibitory, or — in some views — storage
RuBP from the catalytic sites. Activase is also required in some plants because, in darkness,
RuBisCO is inhibited by a competitive inhibitor synthesized by these plants, a substrate analog
2-Carboxy-D-arabitinol 1-phosphate. CA1P binds tightly to the active site of carbamylated
RuBisCO and inhibits catalytic activity. In the light, RuBisCO activase also promotes
the release of CA1P from the catalytic sites. After the CA1P is released from RuBisCO, it
is rapidly converted to a non-inhibitory form by a light-activated CA1P-phosphatase. Finally,
once every several hundred reactions, the normal reactions with carbon dioxide or oxygen
are not completed, and other inhibitory substrate analogs are formed in the active site. Once
again, RuBisCO activase can promote the release of these analogs from the catalytic sites
and maintain the enzyme in a catalytically active form. The properties of activase limit
the photosynthetic potential of plants at high temperatures. CA1P has also been shown
to keep RuBisCO in a conformation that is protected from proteolysis. At high temperatures,
RuBisCO activase aggregates and can no longer activate RuBisCO. This contributes to the
decreased carboxylating capacity observed during heat stress.
Regulation by ATP/ADP and stromal reduction/oxidation state through the activase
The removal of the inhibitory RuBP, CA1P, and the other inhibitory substrate analogs
by activase requires the consumption of ATP. This reaction is inhibited by the presence
of ADP, and, thus, activase activity depends on the ratio of these compounds in the chloroplast
stroma. Furthermore, in most plants, the sensitivity of activase to the ratio of ATP/ADP is modified
by the stromal reduction/oxidation state through another small regulatory protein, thioredoxin.
In this manner, the activity of activase and the activation state of RuBisCO can be modulated
in response to light intensity and, thus, the rate of formation of the ribulose 1,5-bisphosphate
substrate. Regulation by phosphate
In cyanobacteria, inorganic phosphate participates in the co-ordinated regulation of photosynthesis.
Pi binds to the RuBisCO active site and to another site on the large chain where it can
influence transitions between activated and less active conformations of the enzyme. Activation
of bacterial RuBisCO might be particularly sensitive to Pi levels, which can act in the
same way as RuBisCO activase in higher plants. Regulation by carbon dioxide
Since carbon dioxide and oxygen compete at the active site of RuBisCO, carbon fixation
by RuBisCO can be enhanced by increasing the carbon dioxide level in the compartment containing
RuBisCO. Several times during the evolution of plants, mechanisms have evolved for increasing
the level of carbon dioxide in the stroma. The use of oxygen as a substrate appears to
be a puzzling process, since it seems to throw away captured energy. However, it may be a
mechanism for preventing overload during periods of high light flux. This weakness in the enzyme
is the cause of photorespiration, such that healthy leaves in bright light may have zero
net carbon fixation when the ratio of O 2 to CO
2 reaches a threshold at which oxygen is fixed instead of carbon. This phenomenon is primarily
temperature-dependent. High temperature decreases the concentration of CO
2 dissolved in the moisture in the leaf tissues. This phenomenon is also related to water stress.
Since plant leaves are evaporatively cooled, limited water causes high leaf temperatures.
C4 plants use the enzyme PEP carboxylase initially, which has a higher affinity for CO
2. The process first makes a 4-carbon intermediate compound, which is shuttled into a site of
C3 photosynthesis then de-carboxylated, releasing CO
2 to boost the concentration of CO 2, hence the name C4 plants.
Crassulacean acid metabolism plants keep their stomata closed during the day, which conserves
water but prevents the light-independent reactions from taking place, since these reactions require
CO 2 to pass by gas exchange through these openings.
Evaporation through the upper side of a leaf is prevented by a layer of wax.
Genetic engineering Since RuBisCO is often rate-limiting for photosynthesis
in plants, it may be possible to improve photosynthetic efficiency by modifying RuBisCO genes in plants
to increase catalytic activity and/or decrease oxygenation rates. This could improve biosequestration
of CO 2 and be an important climate change strategy.
Approaches under investigation include transferring RuBisCO genes from one organism in another
organism, increasing the level of expression of RuBisCO subunits, expressing RuBisCO small
chains from the chloroplast DNA, and altering RuBisCO genes to increase specificity for
carbon dioxide or otherwise increase the rate of carbon fixation.
One avenue is to introduce RuBisCO variants with naturally high specificity values such
as the ones from the red alga Galdieria partita into plants. This may improve the photosynthetic
efficiency of crop plants, although possible negative impacts have yet to be studied. Advances
in this area include the replacement of the tobacco enzyme with that of the purple photosynthetic
bacterium Rhodospirillum rubrum. In 2014, two transplastomic tobacco lines with functional
Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 were created by replacing
the Rubisco with the large and small subunit genes of the Se7942 enzyme, in combination
with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal
protein, CcmM35. Both increased CO 2 fixation rates.
A recent theory explores the trade-off between the relative specificity (i.e., ability to
favour CO 2 fixation over O
2 incorporation, which leads to the energy-wasteful process of photorespiration) and the rate
at which product is formed. The authors conclude that RuBisCO may actually have evolved to
reach a point of 'near-perfection' in many plants, reaching a compromise between specificity
and reaction rate. Since photosynthesis is the single most effective
natural regulator of carbon dioxide in the Earth's atmosphere, a biochemical model of
RuBisCO reaction is used as the core module of climate change models. Thus, a correct
model of this reaction is essential to the basic understanding of the relations and interactions
of environmental models. A new theory and model of the biochemical reaction of photosynthesis
and the draw-backs of today's most widely used model of photosynthesis is discussed
in volume 29 of Advances in Photosynthesis and Respiration.
History of the term RuBisCO The term "RuBisCO" was coined humorously in
1979, by David Eisenberg at a seminar honouring the retirement of the early, prominent RuBisCO
researcher, Sam Wildman, and also alluded to the snack food trade name "Nabisco" in
reference to Wildman's attempts to create an edible protein supplement from tobacco
leaves. See also
References
Further reading
External links See here for the mechanism of the RuBisCO-catalysed
reaction Rubisco: RCSB PDB Molecule of the Month
The Plant Kingdom's sloth: Protein Spotlight article on the "slothful" enzyme Rubisco