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Rubisco 3


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The Enzyme Function and Structure of Ribulose-1,5-biphosphate Carboxylase-Oxygenase.




            D-Ribulose-1,5-Biphosphate Carboxylase-Oxygenase, also known as RuBisCo, is a crucial enzyme in photosynthesis due to it’s catalytic role in the Calvin cycle via fixation of CO2 to D-Ribulose.  This fixation is RuBisCo’s most noted achievement as it is a method of introducing inorganic carbon into the biosphere.  It is also the most abundant enzyme found in plant leaves and is considered to be the most abundant protein on the planet.

            The structure of RuBisCo varies between two major forms depending on plant or autotrophic bacteria species.  Form 1, a holoenzyme found in higher plants, algae, and cyanobacteria, consists of eight large protein subunits (about 55 kDa) and eight small protein subunits (about 12-13 kDa) and has a height and diameter of about 10nm and 12.5nm respectively.  These large subunits are arranged in a hollow octamer surrounded by two layers of the smaller subunits for an overall hexadecamer structure.  Form 2 can be found in some photosynthetic non-sulfur bacteria and consists of only the two large units (about 50 kDa each).  In Form 1 of RuBisCo, the large and small subunits of the enzyme lock symmetrically to form rings and, ultimately, a barrel-like structure complete with a cavity passing through the center of the molecule.

Figure 1: Space-Filling Model of RuBisCo


Rubisco contains a C4 rotation axis which dissects the barrel (not visible in Figure 1).  Rotation of the enzyme about this axis displaces the large and small subunits 90 degrees per rotation.  There are also four C2 rotation axes that contain the central C4 axis.  Due to the sufficient lack of mirror plane symmetry containing the principle axis the enzyme Rubisco has been identified with an overall point group of D4. 

            The active site of Rubisco is formed through the carbamylation of an “activating” CO2 molecule (Note: not the CO2 that is fixated to RuBP) to a designated lysine residue.  This carbamylation forms the binding site for the main metal cofactor Mg2+.  The Mg2+ ion bound to the activation site is coordinated to six oxygen ligands (ie. Coordination Number = 6)--three carbamic oxygens bound to the enzyme by amino acid residues Lys, Glu, and Asp, and three from water.  This coordination forms an octahedral geometry about the Mg2+ ion.

Figure 2: Bound Mg2+ in activation site



This structure, as well as all other intermediate structures of this site, has completely unsymmetrical geometry and is identified with a point group of C1.  This site is further activated for the fixation of CO2 by the binding of the CO2-accepting molecule Ribulose-1,5-Biphosphate (RuBP) and displacement of two water molecules from the central ion.

Figure 3: Addition of RuBP (R2=-CH2-OPO32- ; R1= -CHOH-CH2OPO32-)



The octahedral geometry about the Mg2+ ion aided by the structural support of the carbamate moieties is crucial for the key step of carbon dioxide addition to RuBP due to the geometry of the addition transition state.  As carbon dioxide approaches the active site it first displaces the remaining water molecule ligands attached to the Mg2+ ion.  Partial bonding between RuBP and the carbonyl carbon of carbon dioxide in the resulting transition state results in carbon bonding, tautomerization, and ultimately the formation of a β-ketoacid and carbon fixation.

Figure 4: Mechanism of Carbon Fixation via Rubisco activation

The octahedral geometry of the activation site also aids in the following steps of the Rubisco mechanism, ultimately resulting in the production of two 3-Phosphoglycerate (3PG) molecules which can be recycled back into the Calvin cycle or used in other central metabolic pathways such as glycolysis, gluconeogenesis, and the tri-carboxylic acid cycle.  The release of the resulting 3PG molecules regenerates the active site with the regeneration of the three original water ligands.

            Despite the obvious requirement of Rubisco for the completion of the carbon cycle through the introduction of inorganic carbon into the metabolic pathway in photosynthesis, many issues have arisen over the efficiency of the enzyme.  Firstly, the rate of the carbon fixation is surprisingly slow for an enzyme of such importance, resulting in the consumption of only one or two CO2 molecules per second.  This may be due in part to the decline of CO2 partial pressure at surface levels over history, as well as the incline of O2 production.  The complexity of the reaction structures, the various intermediates required, and the instability of those intermediates also increases the possibility of side reactions and side products that inhibits the production of the 3PG product.  The Rubisco enzyme catalyzes six total complex transformations, not including highly unstable intermediate steps, requiring complicated chemistry that further reduces the overall efficiency.

            The main, and groundbreaking, inefficiency of the Rubisco enzyme is its lack of substrate specificity.  Because of its many similarities to carbon dioxide and its vast natural abundance, atmospheric oxygen (O2) is often mistaken and incorporated into the reaction mechanism at an alarming rate of 9:1 carbon dioxide fixation to oxygen addition.  The addition of oxygen reduces the efficiency of photosynthesis by 50% by altering the reaction pathway, resulting in the production of only one 3PG and one 2PG in lieu of two 3PG molecules.  This alarming malfunction of the Rubisco enzyme has led to the theory that explains its vast abundance in organisms that rely on photosynthesis to produce sustenance.  Leaf samples in green plants have shown to have roughly 30% of all protein population to be Rubisco alone, a staggering amount which is explained by the need to account for the inherent mistakes made by the enzyme in order to produce the desired product of 3PG.  These findings plus the puzzling negligence of natural selection to improve upon this obvious inefficiency has produced much interest in the biochemical, inorganic, and plant science communities.  Various studies have been published describing in full the various reaction mechanisms, transition states, intermediates, and energies involved in order to more fully understand the naturally chosen active site and its geometry in terms of its efficiency.  Furthermore, a great interest has arisen in the field of genetic and chemical engineering towards Rubisco, where studies hope to improve upon the active site synthetically and reduce the inefficiencies inherent to the enzyme. 





Babu Kannappan and Jill E. Gready Redefinition of Rubisco Carboxylase Reaction Reveals Origin of Water for Hydration and New Roles for Active-Site ResiduesJ. Am. Chem. Soc., 2008, 130 (45), pp 15063–15080 Publication Date (Web): October 15, 2008 (Article) DOI: 10.1021/ja803464a


Harald Mauser,William A. King,Jill E. Gready,*,‡ and T. John Andrews§  CO2 Fixation by Rubisco: Computational Dissection of the Key Steps of Carboxylation, Hydration, and C-C Bond Cleavage J. Am. Chem. Soc. 2001, 123, 10821-10829




V. L. Tsuprun ‘, E.J. Boekema’, T.G. Samsonidze’ and A.V. Pushkin. Electron Microscopy of the Complexes of Ribulos-1,5-biphosphate Carboxylase (Rubisco) and Rubisco Subunit-Binding Protein from Pea Leaves.Volume 289, number 2, 205-209 FEBS 10136 September 199 I






Tiaz, Lincoln & Zeiger, Eduardo. Rubisco: A Model Enzyme for Studying Structure and Function. “Plant Physiology, Fourth Edition”



Wikipedia. Rubisco.  <>  May 16, 2010