User:Littlebird9/sandbox

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Reaction Pathway and Current Mechanistic Understanding[edit]

Figure 4: Two possible mechanisms for carbon fixation in RuBisCO. The overall reaction mechanism and the sequential carboxylation and hydration mechanism is reproduced from that described in Taylor and Anderson 1997. Only the concerted carboxylation and hydration mechanism is reproduced from Cleland et. al. 1998. Reactant and products are boxed and have carbon molecules numbered.

Overview[edit]

When Rubisco facilitates the attack of CO2 at the C2 carbon of RuBP and subsequent bond cleavage between the C3 and C2 carbon, 2 molecules of glycerate-3-phosphate are formed. In order to do this the necessary reaction steps are enolisation, carboxylation, hydration, bond cleavage, and stereospecific protonation (Figure 4).[1] The magnesium ion, Mg2+, in the active site is central to the coordination of these steps in the carbon fixation catalysis in RuBisCO.[1][2][3]

Binding RuBP[edit]

After Rubisco is activated by the carbamylation of the ε-amino group of active-site Lys201[4], described above, it is stabilized by coordination with the Mg2+. Additionally, water molecules are exchanged with substrates as ligands over the course of the reaction, while Asp203 and Glu204 ligate the Mg2+ ion throughout the reaction (Figure 5). The substrate RuBP displaces two of the three initial water molecules ligated to Mg2+ in order to bind to the active site.[1][5][6]

Enolisation[edit]

Enolisation of RuBP is the conversion of the ketone form of RuBP to an enediol(ate) with a double bond between C3 and C2 (Figure 4). For enolisation to occur RuBP is deprotonated at C3 by a base. The enzyme base in this step has been debated [5][7], but the steric constraints observed in crystal structures have made Lys201 the most likely candidate.[1] Specifically, the carbamate oxygen on Lys201 that is not coordinated with the Mg ion deprotonates the C3 carbon of RuBP to form a 2,3-enediolate.[5][6] Computational methods probing the energetics of reaction stress the importance of the carbamylated Lys201 in enolisation.[8][9] Lys175 or His294 may act as an acid for the protonation of the oxygen of C2 after this step[1][10] so that the CO2 substrate will attack the C2 carbon rather than the C3 carbon in subsequent carboxylation or oxygenation. The C2 and C3 centers are moulded in a cis out of plane conformation in the transition structure around the C2-C3 bond, which provides the necessary activation for the reaction to proceed[11][12]

Carboxylation and Hydration[edit]

Figure 5: A 3D image of the active site of spinach RuBisCO complexed with the inhibitor 2-Carboxyarabinitol-1,5-Bisphosphate, CO2, and Mg2+. (PDB: 1IR1; Ligand View [CAP]501:A)

Carboxylation of the 2,3-enediolate results in the intermediate 3-keto-2′-carboxyarabinitol-1,5-bisphosphate and Lys334 is positioned to facilitate the addition of the CO2 substrate as it replaces the third Mg2+-coordinated water molecule and add directly to the enediol (Figure 5). No Michaelis complex is formed in this process.[1][7] Hydration of this ketone results in an additional hydroxy group on C3, forming a gem-diol intermediate.[5][10] Carboxylation and hydration have been proposed as either a single concerted step[5] or as two sequential steps.[10] Concerted mechanism is supported by the proximity of the water molecule to C3 of RuBP in multiple crystal structures. Within the spinach structure, other residues are well placed to aid in the hydration step as they are within hydrogen bonding distance of the water molecule.[1]

C-C bond Cleavage and Stereo Specific Protonation[edit]

Once the hydroxy group is added to C3 in hydration, the gem-diol intermediate can be cleaved at the C2-C3 bond to form one molecule of glycerate-3-phosphate and a negatively charge carboxylate ion intermediate.[1] The electrons from an oxygen on C3 form a double bond with C3 and the C2 carbon leaves as a carbanion.[13][14][15] Stereo specific protonation of C2 of this carbanion results in another molecule of glycerate-3-phosphate. This step is thought to be facilitated by Lys175 or potentially the carbamylated Lys201.[1]

  1. ^ a b c d e f g h i Andersson, Inger (May 2008). "Catalysis and regulation in Rubisco". Journal of Experimental Botany. 59 (7): 1555–1568.
  2. ^ Erb, Tobias; Zarzycki, Jan (February 2018). "A short history of RubisCO: the rise and fall (?) of Nature's predominant CO2 fixing enzyme". Current Opinion in Biotechnology. 49: 100–107.
  3. ^ Schneider, Gunter; Lundqvis, Tomas (5 July 1991). "Crystal Structure of Activated Ribulose- 1,5-bisphosphate Carboxylase Complexed with Its Substrate, Ribulose- 1,5-bisphosphate*". The Journal of Biological Chemistry. 266 (19): 12604–12611.
  4. ^ Lorimer, G; Miziorko, H (1980). "Carbamate Formation on the c-Amino Group of a Lysyl Residue as the Basis for the Activation of Ribulosebisphosphate Carboxylase by C02 and Mg2+". Biochemistry. 19: 5321–5328. doi:10.1021/bi00564a027.
  5. ^ a b c d e Cleland, W; Lorimer, G (1998). "Mechanism of Rubisco: The Carbamate as General Base". American Chemical Society Chemical Review. 98 (2): 549−561. doi:10.1021/cr970010r.
  6. ^ a b Andersson, I; Knight, S; Schneider, G; Lindqvist, Y; Lindqvist, T; Brändén, CI; Lorimer, GH (1989). "Crystal structure of the active site of ribulose-bisphosphate carboxylase". Nature. 337: 229–234.
  7. ^ a b F, Hartman; M, Harpel (1994). "STRUCTURE, FUNCTION, REGULATION, AND ASSEMBLY OF D-RIBULOSE-l,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE". Annual Review of Biochemistry. 63: 197–234.
  8. ^ Mauser, H; Andrews, T (2001). "CO2 Fixation by Rubisco:  Computational Dissection of the Key Steps of Carboxylation, Hydration, and C−C Bond Cleavage". Journal of the American Chemical Society. 123 (44): 10821–10829. doi:10.1021/ja011362p.
  9. ^ King, W; Gready, J; Andrews, T (1998). "Quantum Chemical Analysis of the Enolization of Ribulose Bisphosphate:  The First Hurdle in the Fixation of CO2 by Rubisco". Biochemistry. 37 (44): 15414–15422. doi:10.1021/bi981598e.
  10. ^ a b c Taylor, TC; Andersson, I (1997). "The structure of the complex between rubisco and its natural substrate ribulose-1,5-bisphosphate". Journal of Molecular Biology. 265: 432–444.
  11. ^ Topia, O; Andres, J (July 1993). "A theoretical study of the singlet-triplet energy gap dependence upon rotation and pyramidalization for 1,2-dihydroxyethylene: a simple model to study the enediol moiety in Rubisco's substrate". The Journal of Physical Chemistry. 97 (30): 7888–7893. doi:10.1021/j100132a016.
  12. ^ Tapia, O; Andres, Juan (23 January 2002). "Enzyme catalysis: Transition structures and quantum dynamical aspects: Modeling rubisco's oxygenation and carboxylation mechanisms". International Journal of Quantum Chemistry.
  13. ^ Mauser, H; King, W; Gready, J; Andrews, T (2001). "CO2 Fixation by Rubisco: Computational Dissection of the Key Steps of Carboxylation, Hydration, and C-C Bond Cleavage". Journal of the American Chemical Society. 123: 10821–10829. doi:10.1021/ja011362p.
  14. ^ Cummins, P; Gready, J (2018). "Revised Mechanism of Carboxylation of Ribulose-1,5- Biphosphate by Rubisco from Large Scale Quantum Chemical Calculations". Journal of Computational Chemistry. 39: 1656–1665.
  15. ^ B, Kannappan; J, Gready (2008). "Redefinition of Rubisco Carboxylase Reaction Reveals Origin of Water for Hydration and New Roles for Active-Site Residues". Journal of the American Chemical Society. 130 (45): 15063–15080. doi:10.1021/ja803464a.
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