Rubisco (PDB ID:1RXO) from Spinacia oleracea
Created by: Maggie Schwartz
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly called rubisco (PDB: 1RXO) is an enzyme often found in the stroma of chloroplasts
that catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP) (8). Rubisco is thought to be most abundant enzyme on earth, and responsible for the first step of photosynthesis: the incorporation of atmospheric CO2 into the biosphere (3). However, rubisco is both slow and inefficient due to a competing reaction with atmospheric O2 where it can act as an oxygenase (2). Consequently, the first step of photosynthesis is the rate-limiting step as well (2). Rubisco has been isolated from many different species, including Spinacia oleracea, where its structure was elucidated through crystallization of the enzyme with a calcium ion (instead of magnesium) and in complex with RuBP (11).
The inefficiency of rubisco is significant in limiting the growth of plants (4). Accordingly, rubisco has been the subject of heavy research for over 50 years as scientists attempt to develop a more efficient enzyme (4). Rubisco is not only relevant agriculturally, but environmentally as well, especially in light of the importance of CO2 as a greenhouse gas (2). Through this research, much has been uncovered structurally and proposed mechanistically about rubisco.
Rubisco is a relatively large protein. In Spinacia oleracea, it has a molecular weight of 269 kD (12). Its theoretical isoelectric point is 6.09, which is important mechanistically in allowing for spontaneous carbamylation in alkaline conditions (12, 5). Across a diverse range of species, the secondary and tertiary folds of subunits, along with the primary sequences of segments associated with the active site are extremely well conserved (4). In comparison of the large subunit to that of Nicotiana tabacum (tobacco), rubisco has a very high Z score of 58.9, showing nearly identical subunit structure (6). Analysis of primary structure showed high homology to a variety of plants, including rubisco from Salix traindra (almond willow), showing an impressive E value of 0.0 (1). The various domains of rubisco are highly conserved as well with E values for each subunit ranging from 0.0 to 1.58e-54 (9). Due to all these structural similarities and its importance in photosynthesis, the large subunits rubisco are likely evolutionarily ancient (10).
In Spinacia oleracea, rubisco is a hexadecamer
composed of eight large (L) and eight small (S) subunits in an L8S8 arrangement (11). The composition of secondary structure varies between subunits, with S subunits at 21% helical and 23% beta sheet, and the L subunits at 41% helical and 16% beta sheet (7). The catalytic (L) subunit is an eight-stranded parallel alpha/beta barrel (2). The residues that create the active site are found in the loops that connect beta-strands with alpha-helices on the carboxy-terminal side of the barrel (11). Specifically, in loop 2 between strand 2 and helix 2 there are three important ligands that serve to coordinate a magnesium ion needed for catalysis (Lys201, Asp 203, Glu204) (11). Corresponding to the eight L subunits, there are eight total active sites for each enzyme of rubisco. Rubisco exists in an open (active) or closed (inactive) form, and closing of the active site occurs by movements of loop 6 
(residues 332-338) of the alpha/beta barrel, the carboxy-terminus of the L subunit, loops from the amino-terminal domain of the adjacent L subunit, and a shift in the associated S subunit (11). Once over the active site, the Lys334 from loop 6 interacts closely with the substrate, reaching into the active site and making contact with the P1 phosphate group of RuBP (11). The trigger for closing the active site is the distance between the P1 and P2 phosphate groups of RuBP (4). If the distance between these two groups is less than 9.1 Angstroms, then the active site closes; if it is greater than 9.4 Angstroms, then it remains open (4). The reaction mechanism is proposed to occur through electron and proton transfers entirely within the active site with no major structural transitions (4).
Before catalysis can take place, rubisco must be in its active form. This involves two processes, the first of which is carbamylation of Lys201. This occurs through the addition of CO2 to the sigma-NH2 group of Lysine (5). This CO2 that has been added will not be used in the later carboxylation of RuBP (5). Carbamylation occurs spontaneously in slightly alkaline conditions with a pH of about 8 (5). Though the rubisco has been carbamylated, it is still not activated. The second process of activation is the association of a Mg 2+ ion at the now carbamylated Lys201 (5). The negative charge introduced on Lysine can serve as a ligand for Mg2+ (8). Once activated, rubisco fixates atmospheric CO2 to RuBP ultimately forming two 3-phosphoglycerates (3PG) (11). First, an enediol intermediate is thought to be formed when a proton is pulled off C-3 of RuBP by His327, Lys334, Ser379, or the carbamate nitrogen from Lys201 (8). Then the chemical pathway proceeds through an enzyme-bound intermediate called 2-carboxy-3-keto-arabinitol, which forms when CO2 is added to the enediol intermediate from RuBP (5). Once RuBP is carboxylated, hydrolysis of the C2-C3 bond generates two 3PGs 
(5). These 3PGs proceed to the Calvin-Benson cycle where they are eventually transformed into hexoses through a series of reactions (5). 
Interestingly, though it is the substrate of the enzyme, RuBP is also a strong inhibitor of rubisco as it binds much more tightly to the inactive form than the active form (5). Rubisco activase acts as a regulatory protein for rubisco. When rubisco activase binds to the inactive form of rubisco, it promotes the release of RuBP through an ATP-dependent reaction (5). Rubisco activase is regulated indirectly by light, and correspondingly, rubisco is made more active when light is present (5). In fact, light activates rubisco in several ways. Changes in pH occur in chloroplasts as light causes protons to be pumped into the thylakoid lumen, leading to rises in stromal pH values (5). This alkaline environment in the stroma promotes carbamylation of Lys201. As protons flow from stroma to thylakoid lumen, Mg2+ ions flow from vesicles in the opposite direction simultaneously in order to balance changes in electrical charge across the membrane (5). Since Mg2+ is required for catalysis, this ion flow caused indirectly by light also leads to activation of rubisco. The activity of rubisco is also regulated by a nocturnal inhibitor 2-carboxy-D-arabinitol 1-phosphate (CA1P) that binds to carbamylated rubisco and increases in concentration in the dark (8). 
As its name suggests, ribulose-1,5-bisphosphate carboxylase/oxygenase can also act as an oxygenase in the presence of atmospheric O2. In fact, the ratio of carboxylase to oxygenase activity is about 3 or 4 to 1 (5). This process results in the synthesis of products 3-phosphoglycerate and phosphoglycolate (4). In order to make use of phosphoglycolate, it must be re-circulated by photorespiration, a series of reactions that requires energy in the form of ATP (2). Oxygenase activity causes a constant drain on the RuBP supply, and can decrease carbon fixation efficiency by up to 50% (2). 
Much more is known about the structures and functions of the L subunits than the S subunits, and consequently the role of S subunits in rubisco activity is a point of current research. Two forms of rubisco exist in some prokaryotes (10). Form-I resembles that found in Spinacia oleracea with an L8S8 structure, while Form-II has only L subunits (10). In comparing the activity of these two forms, it has been proposed that S subunits may aid in the assembly of rubisco as well as the concentration of it, allowing more rubisco to exist in a given cell (10). Additionally, the S subunits may increase specificity for CO2, leading to better overall carbon fixation efficiency (10). The precise role of the S subunit is an area that remains to be researched in the future to perhaps achieve better efficiency for rubisco (10).