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CH2O-® S-Phosphoglycerate

CH2O-® Glyceraldehyde S-phosphate

FIGURE 20-4 The three stages of CO2 assimilation in photosynthetic organisms. Stoichiome-tries of three key intermediates (numbers in parentheses) reveal the fate of carbon atoms entering and leaving the cycle. As shown here, three CO2 are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate. This cycle is the photosynthetic carbon reduction cycle, or the Calvin cycle.

FIGURE 20-4 The three stages of CO2 assimilation in photosynthetic organisms. Stoichiome-tries of three key intermediates (numbers in parentheses) reveal the fate of carbon atoms entering and leaving the cycle. As shown here, three CO2 are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate. This cycle is the photosynthetic carbon reduction cycle, or the Calvin cycle.

Stage 1: Fixation of CO2 into 3-Phosphoglycerate An important clue to the nature of the CO2-assimilation mechanisms in photosynthetic organisms came in the late 1940s. Calvin and his associates illuminated a suspension of green algae in the presence of radioactive carbon dioxide (14CO2) for just a few seconds, then quickly killed the cells, extracted their contents, and with the help of chromatographic methods searched for the metabolites in which the labeled carbon first appeared. The first compound that became labeled was 3-phos-phoglycerate, with the 14C predominantly located in the carboxyl carbon atom. These experiments strongly suggested that 3-phosphoglycerate is an early intermediate in photosynthesis. The many plants in which this three-carbon compound is the first intermediate are called C3 plants, in contrast with the C4 plants described below.

The enzyme that catalyzes incorporation of CO2 into an organic form is ribulose 1,5-bisphosphate carbox-ylase/oxygenase, a name mercifully shortened to ru-bisco. As a carboxylase, rubisco catalyzes the covalent attachment of CO2 to the five-carbon sugar ribulose 1,5-bisphosphate and cleavage of the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO2 in its carboxyl group (Fig. 20-4). The enzyme's oxygenase activity is discussed in Section 20.2.

Plant rubisco, the crucial enzyme in the production of biomass from CO2, has a complex structure (Fig. 20-5a), with eight identical large subunits (Mr 53,000; encoded in the chloroplast genome, or plastome), each containing a catalytic site, and eight identical small sub-units (Mr 14,000; encoded in the nuclear genome) of uncertain function. The rubisco of photosynthetic bacteria is simpler in structure, having two subunits that in many respects resemble the large subunits of the plant enzyme (Fig. 20-5b). This similarity is consistent with the endosymbiont hypothesis for the origin of chloro-plasts (p. 35). The plant enzyme has an exceptionally low turnover number; only three molecules of CO2 are fixed per second per molecule of rubisco at 25 °C. To achieve high rates of CO2 fixation, plants therefore need large amounts of this enzyme. In fact, rubisco makes up almost 50% of soluble protein in chloroplasts and is probably one of the most abundant enzymes in the biosphere.

Central to the proposed mechanism for plant ru-bisco is a carbamoylated Lys side chain with a bound Mg2+ ion. The Mg2+ ion brings together and orients the reactants at the active site (Fig. 20-6) and polarizes the CO2, opening it to nucleophilic attack by the five-carbon enediolate reaction intermediate formed on the enzyme (Fig. 20-7). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate.

FIGURE 20-5 Structure of ribulose 1,5-bisphosphate carboxylase (rubisco). (a) Top and side view of a ribbon model of rubisco from spinach (PDB ID 8RUC). The enzyme has eight large subunits (blue) and eight small ones (gray), tightly packed into a structure of Mr 550,000. Rubisco is present at a concentration of about 250 mg/mL in the chloroplast stroma, corresponding to an extraordinarily high concentration of active sites (~4 mM). Amino acid residues of the active site are shown in yellow, Mg2+ in green. (b) Ribbon model of rubisco from the bacterium Rhodospirillum rubrum (PDB ID 9RUB). The subunits are in gray and blue. A Lys residue at the active site that is carboxylated to a carbamate in the active enzyme is shown in red. The substrate, ribulose 1,5-bisphosphate, is yellow; Mg2+ is green.

FIGURE 20-5 Structure of ribulose 1,5-bisphosphate carboxylase (rubisco). (a) Top and side view of a ribbon model of rubisco from spinach (PDB ID 8RUC). The enzyme has eight large subunits (blue) and eight small ones (gray), tightly packed into a structure of Mr 550,000. Rubisco is present at a concentration of about 250 mg/mL in the chloroplast stroma, corresponding to an extraordinarily high concentration of active sites (~4 mM). Amino acid residues of the active site are shown in yellow, Mg2+ in green. (b) Ribbon model of rubisco from the bacterium Rhodospirillum rubrum (PDB ID 9RUB). The subunits are in gray and blue. A Lys residue at the active site that is carboxylated to a carbamate in the active enzyme is shown in red. The substrate, ribulose 1,5-bisphosphate, is yellow; Mg2+ is green.

FIGURE 20-6 Central role of Mg2+ in the catalytic mechanism of rubisco. (Derived from PDB ID 1RXO) Mg2+ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on Lys201; two in the carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, CO2. A water molecule occupies the Co2-binding site in this crystal structure. (Residue numbers refer to the spinach enzyme.)

FIGURE 20-6 Central role of Mg2+ in the catalytic mechanism of rubisco. (Derived from PDB ID 1RXO) Mg2+ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on Lys201; two in the carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, CO2. A water molecule occupies the Co2-binding site in this crystal structure. (Residue numbers refer to the spinach enzyme.)

Rubisco

AsP.

Rubisco

AsP.

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