The Calvin cycle takes place in the stroma of chloroplasts, the photosynthetic organelles. In this vital process, carbon dioxide gas is trapped in an organic molecule, 3-phosphoglycerate, which has many biochemical fates.

The Calvin cycle has three stages (Figure 23.1):

Keep in mind that, owing to the stoichiometry of the conversion of six molecules of CO₂ into a hexose sugar, to synthesize a glucose molecule or any other hexose sugar, the three stages must take place six times. The regeneration process is a complicated one—hence the dotted arrow and simplification of the process depicted in Figure 23.1; the details will be revealed as we move through the process. For simplicity’s sake, we will consider the reactions one molecule at a time or three molecules at a time.

429

The first stage in the Calvin cycle is the fixation of CO₂. This highly exergonic reaction (ΔG°′ = −51.9 kJ mol⁻¹, or −12.4 kcal mol⁻¹) is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (usually called rubisco), an enzyme located on the stromal surface of the thylakoid membranes of chloroplasts. This reaction is the rate-limiting step in hexose synthesis.

Carbon fixation begins with the conversion of ribulose 1,5-bisphosphate into a highly reactive enediolate intermediate. The CO₂ molecule condenses with the enediolate intermediate to form an unstable six-carbon compound, which is rapidly hydrolyzed to two molecules of 3-phosphoglycerate:

Thus, carbon dioxide, a waste product of cellular respiration, is reintroduced to the biochemical world. Plants that use only the Calvin cycle to fix carbon dioxide are called C₃ plants, because the three-carbon molecule 3-phosphoglycerate is formed immediately after the rubisco reaction.

Rubisco in chloroplasts consists of eight large (L, 55-kDa) subunits and eight small (S, 13-kDa) ones (Figure 23.2). Each L chain contains a catalytic site and a regulatory site. The S chains enhance the catalytic activity of the L chains. This enzyme is very abundant in chloroplasts, constituting more than 16% of their total protein. In fact, rubisco is the most abundant enzyme in plants and probably the most abundant protein in the biosphere. Large amounts are required because rubisco is an inefficient enzyme; its maximal catalytic rate is only 3 s⁻¹. Rubisco also requires a bound CO₂ for catalytic activity in addition to the substrate CO₂. The bound CO₂ forms a carbamate with lysine 201 of the large subunit. The carbamate binds to a Mg²⁺ ion that is required for catalytic activity. We will see the regulatory significance of this requirement later.

In the absence of CO₂, rubisco binds to its substrate, ribulose 1,5-bisphosphate, so tightly that its enzyme activity is inhibited. The enzyme rubisco activase uses ATP to induce structural changes in rubisco that allow release of the bound substrate and formation of the required carbamate. The ATP requirement of rubisco activase coordinates rubisco activity with the light reactions.

430

In the second stage of the Calvin cycle, the 3-phosphoglycerate products of rubisco are converted into a hexose phosphate. The steps in this conversion (Figure 23.3) are like those of the gluconeogenic pathway, except that glyceraldehyde 3-phosphate dehydrogenase in chloroplasts, which generates glyceraldehyde 3-phosphate (GAP) in the process of hexose formation, is specific for NADPH rather than NADH. The hexose phosphate product of the Calvin cycle exists in three isomeric forms: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, together called the hexose monophosphate pool. Recall that these isomers are readily interconvertible. Alternatively, the glyceraldehyde 3-phosphate can be transported to the cytoplasm and metabolized to fructose 6-phosphate and glucose 1-phosphate by using the gluconeogenic pathway and the enzyme phosphoglucomutase. Fructose 6-phosphate and glucose 1-phosphate are used for sucrose synthesis (Figure 23.8). These reactions and the reaction catalyzed by rubisco bring CO₂ to the oxidation level of a hexose, converting CO₂ into a chemical fuel at the expense of the NADPH and ATP that were generated in the light reactions.

The third and final stage of the Calvin cycle is the regeneration of ribulose 1,5-bisphosphate, the acceptor of CO₂ in the first stage. This regeneration is not as simply done as the regeneration of oxaloacetate in the citric acid cycle. The challenge is to construct a five-carbon sugar from a six-carbon member of the hexose monophosphate pool and three-carbon molecules, such as glyceraldehyde 3-phosphate. A transketolase, which we will encounter again in the pentose phosphate pathway, and an aldolase, an enzyme in glycolysis and gluconeogenesis, have major roles in the rearrangement of the carbon atoms. With these enzymes, the construction of the five-carbon sugar proceeds as shown in Figure 23.4. The sum of these reactions is

This series of reactions completes the Calvin cycle (Figure 23.5). Figure 23.5 presents the required reactions with the proper stoichiometry to convert three molecules of CO₂ into one molecule of dihydroxyacetone phosphate (DHAP). However, two molecules of DHAP are required for the synthesis of a member of the hexose monophosphate pool. Consequently, the cycle as presented must take place twice to yield a hexose monophosphate. The outcome of the Calvin cycle is the generation of a hexose and the regeneration of the starting compound, ribulose 1,5-bisphosphate. In essence, ribulose 1,5-bisphosphate acts catalytically, similarly to oxaloacetate in the citric acid cycle.

What is the energy expenditure for synthesizing a hexose? Six rounds of the Calvin cycle are required, because one carbon atom is reduced in each round. Twelve molecules of ATP are expended in phosphorylating 12 molecules of 3-phosphoglycerate to 1,3-bisphosphoglycerate, and 12 molecules of NADPH are consumed in reducing 12 molecules of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. An additional 6 molecules of ATP are spent in regenerating ribulose 1,5-bisphosphate. The balanced equation for the net reaction of the Calvin cycle is then

431

432

Thus, 3 molecules of ATP and 2 molecules of NADPH are consumed in incorporating a single CO₂ molecule into a hexose such as glucose or fructose. Biochemically, the synthesis of glucose from CO₂ is energetically expensive, but the ultimate energy source—sunlight—is abundant.

What are the fates of the carbon atoms fixed and processed by the enzymes of the Calvin cycle? These molecules are used in a variety of ways, but there are two prominent fates: the synthesis of starch and sucrose, storage forms of carbohydrates. Starch, like its animal counterpart glycogen, is a polymer of glucose residues. Starch comes in two forms: amylose, which consists of glucose molecules joined together by α-1,4 linkages only; and amylopectin, which is branched because of the presence of α-1,6 linkages. Starch is synthesized and stored in chloroplasts.

In contrast, sucrose (common table sugar), a disaccharide, is synthesized in the cytoplasm. Plants lack the ability to transport hexose phosphates across the chloroplast membrane, but an abundant triose phosphatephosphate antiporter mediates the transport of triose phosphates from chloroplasts to the cytoplasm in exchange for phosphate. Fructose 6-phosphate is formed from these triose phosphates in the cytoplasm and joins the glucose unit of cytoplasmic UDP-glucose (activated glucose) to form sucrose 6-phosphate (Figure 23.8). Sucrose 6-phosphate is then hydrolyzed by sucrose 6-phosphatase to yield sucrose. Sucrose is transported from the leaves as a component of sap to the nonphotosynthetic parts of the plant, such as the roots, and is stored in many plant cells, as in sugar beets and sugar cane (Figure 23.9).

434