Rubisco - reaction mechanism

The enzyme ribulose-1,5-bisphosphate carboxylase (rubisco) catalyzes the addition of gaseous carbon dioxide to ribulose-1,5-bisphosphate (RuBP). Product of the reaction are two molecules of 3-phosphoglyceric acid.

C5O3H8(PO42-)2 + CO2 -> 2 C3O3H4PO42-

For the reaction to occur it is neccessary that the protein is activated by the covalent binding of a molecule of carbon dioxide. This CO2 forms a carbamate with the terminal amino group of lysine201. The carbamate itself is labile, it is stabilized in the enzyme by a magnesium ion.

The figures to follow may be viewed in an interactive manner: using the mouse the molecules can be moved; clicking the boxes highlights the described features. Only the first figure is backed by experimental data. Hydrogen atoms (which are not visible in a X-ray data set) were added by a molecular modelling program. The reaction steps were also modeled - there is no claim as to the absolute positions of the atoms concerned. The figures are merely meant to visualize the reaction mechanism.

colour scheme:   C H O N P Mg

1

Within the active site of the enzyme the magnesium atom is fixed by amino acids 201 to 204.
Lys201 with the carbamate group
Asp202
Asp203
Glu204
The magnesium ion is coordinated by six oxygen atoms:
by three amino acids
by one water
twice by the substrate RuBP
  - by the keto oxygen off C2
  - by the hydroxyl ogygen off C3
The oxygen atoms form a somewhat distorted octahedron around the magnesium (distances in Å).

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2

The first step on the reaction pathway is the tautomerisation of RuBP to the enediolate. In this reaction the hydrogen at C3 (fig. 1) is bound to the carbamate group of lys201 . The oxygen atom at C2 receives the negative charge of the carbamate oxygen. C3 has been sp3 hybridized with tetrahedral ligands before. During this step it is hybridized to sp2 with a trigonal configuration. The ligands of the C2-C3 double bond are in cis (turning the molecule with the mouse will show the planar setting).

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3

The next step is the binding of a molecule of CO2 , which displaces a water molecule. The CO2 itself is polarized by the vicinity of the metal ion and thus can perform an electrophilic attack on the enediolate. This again is accompanied by a change of positions of hydrogen atoms: the hydrogen of the C3-hydroxyl (fig. 2) is translocated to a nitrogen atom on his294. The hydrogen previously bound to the carbamate on lys201 is transferred to the enediolate oxygenon C2 of the RuBP molecule . The new covalent bond between CO2 and RuBP results in a branched C6 sugar.

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4

The covalent bond between CO2 and RuBP-C2 forces C2 temporarily to a sp3 tetrahedral conformation (a change in hybridization pattern results in diffenrent bonding angels, which causes the side chains to move).
The next step is the hydroxylation of C3. A water molecule looses a hydrogen atom to his327 and the oxygen attacks C3.

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5

The newly formed intermediate with two oxygen atoms bound to C3 is deprotonated by the aid of lys201 . The other oxygen atom on C3 is held in a deprotonated state by his294 . In this very moment the electron configuration of C3 is less than optimal. It is brought to a normal state by a cleavage of the bond between C3 and C2, so it regains a sp2 hybridization characteristic of a carboxyl group with planar ligands.

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6

The result is one molecule of 3-phosphoglycerate . In all reactions so far only the carboxyl carbon of this molecule took part. Thus the conformation of it's chiral (middle) carbon atom remained unchanged. If (with aid of the mouse) you turn the molecule so that (with a vertical orientation of the chain, carboxyl end to the top) both the carboxyl carbon and the terminal carbon (bearing the phosphate group) point to the rear, you may find the hydroxyl group of the middle carbon pointing to the right. That means without doubt this molecule is a D-phosphoglycerate. It is released from the enzyme.
The rest of the carboxylated molecule remained with a lone electron pair on C2. Lys175 supplies a proton as a fourth ligand to C2.

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7

This addition leads from the intermediate to the reaction product. The reaction is stereospecific: another molecule of D-3-phosphoglycerate arises .

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Literature: C Taylor & I Andersson, J. Mol. Biol. 265 (1997) 432-444

9-98 © R Bergmann