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How were the steps in carbohydrate synthesis elucidated?

To identify the reactions by which the carbon from CO₂ ends up in carbohydrates, scientists found a way to label CO₂ so they could isolate and identify the compounds formed from it during photosynthesis. In the 1950s, Melvin Calvin, Andrew Benson, and their colleagues used radioactively labeled CO₂ in which some of the carbon atoms were the radioisotope ¹⁴C rather than the normal ¹²C. They were able to trace the chemical pathway of CO₂ fixation (Figure 10.10). The first molecule that appeared in the pathway was a three-carbon sugar phosphate called 3-phosphoglycerate (3PG) (the ¹⁴C is shown in red):

experiment

Figure 10.10A Tracing the Pathway of CO₂

Original Papers: Calvin and his colleagues described their experiments in a series of 26 papers titled “The Path of Carbon in Photosynthesis.” Perhaps the most important was one that showed how labeled CO₂ could be used a tracer:

Benson, A. A., J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas and W. Stepka. 1950. The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products. Journal of the American Chemical Society 72: 1710–1718.

How is CO₂ incorporated into carbohydrate during photosynthesis? What is the first stable covalent linkage that forms with the carbon of CO₂? Melvin Calvin and his colleagues used short exposures to ¹⁴CO₂ to identify the first compound formed from CO₂.

Figure 10.10B work with the data follows on the next page.

work with the data

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Figure 10.10B Tracing the Pathway of CO₂

Original Paper: Benson, A. A. et al. 1950.

To elucidate the sequence of reactions that allow carbon fixation, Melvin Calvin and colleagues exposed suspensions of the green alga Chlorella to ¹⁴CO₂ (see Figure 10.10A). After 3 sec of photosynthesis, the ¹⁴C from ¹⁴CO₂ was found only in 3-phosphoglycerate (3PG), but after 30 sec many compounds were radioactive. Calvin and his colleagues then expanded on these results and were able to determine the exact sequence of reactions and reaction intermediates in the Calvin cycle by exposing the cells to ¹⁴CO₂ for various periods of time.

The first reaction in CO₂ fixation can occur in the dark. To show this, Calvin and his colleagues exposed Chlorella cells to ¹⁴CO₂ under bright lights for 20 min and harvested the cells. Then they repeated the experiment, but following the 20 min of light with varying periods of darkness (30 sec, 2 min, and 5 min). They harvested cells and made chromatograms to identify the labeled compounds, and used a radioactivity detector to quantify the amount of ¹⁴C in each compound. The data are shown in the table.

	Relative amount of radioactivity after:
Compound	20 min light	20 min light + 30 sec dark	20 min light + 2 min dark	20 min light + 5 min dark
3PG	5,500	10,100	10,000	5,200
RuBP	4,900	680	1,850	1,800
Sucrose	13,000	13,500	15,000	14,750

QUESTIONS

Question 1

Plot radioactivity in 3PG versus time. What do the data show?

The data show an initial rise of 3PG (in first 30 sec) because rubisco is initially still active and can catalyze the reaction of CO2 with RuBP to produce 3PG. Between 30 sec and 2 min, the amount of 3PG levels off as rubisco becomes inactive in the dark. After 2 min, the amount of 3PG falls as it enters other pathways (see Figure 10.18).

Question 2

Why did the amount of radioactively labeled RuBP decline after 30 seconds in the dark?

The level of RuBP went down in the dark initially because it was consumed in the reaction catalyzed by rubisco.

Animation 10.3 Tracing the Pathway of CO₂

www.life11e.com/a10.3

A similar work with the data exercise may be assigned in LaunchPad.

Using successively longer exposures to ¹⁴CO₂, Calvin and his colleagues were able to trace the route of ¹⁴C as it moved through a series of compounds, including monosaccharides and amino acids. It turned out the ¹⁴C moved through a cyclical pathway. In this cycle, the CO₂ initially bonds covalently to a five-carbon acceptor molecule. The resulting six-carbon intermediate quickly breaks into two three-carbon molecules. As the cycle repeats, a carbohydrate is produced and the initial CO₂ acceptor is regenerated. This pathway was appropriately named the Calvin cycle.

The initial reaction in the Calvin cycle adds the one-carbon CO₂ to the five-carbon acceptor molecule ribulose 1,5-bisphosphate (RuBP). The product is an intermediate six-carbon compound, which quickly breaks down and forms two molecules of 3PG (Figure 10.11). The intermediate compound is broken down so rapidly that Calvin did not observe radioactive carbon appearing in it first. The enzyme that catalyzes its formation, ribulose bisphosphate carboxylase/oxygenase (rubisco), is the most abundant protein in the world! It constitutes up to 50 percent of all the protein in every plant leaf.

Figure 10.11 RuBP Is the Carbon Dioxide Acceptor CO₂ is added to a five-carbon compound, RuBP. The resulting six-carbon compound immediately splits into two molecules of 3PG.