In this article we will discuss about:- 1. Meaning of Calvin Cycle 2. Features of Calvin Cycle 3. Enzymes.
Meaning of Calvin Cycle:
Like the photosynthetic reactions which are driven by the light energy the reactions which joint hydrogen and CO2 to produce carbohydrate also require energy and the main source of this energy is the assimilatory power (ATP + NADPH2) regenerated in photolysis. Melvin Calvin and his colleagues at a Berkeley, University of California, worked out the reactions by which CO2 is incorporated into organic compounds.
They incubated algal suspensions (Chlorella) in the presence of CO2labelled with 14C (14CO2) for varying durations of time and isolating and identifying the products after specific intervals. They could thus trace the sequence of reactions leading to the formation of the end product. Calvin eventually received the Nobel Prize for his work.
When the photosynthesizing plant was exposed to the 14CO2 for only 5 seconds, 14C appeared in 3-phosphoglyceric acid (3-PGA), which is normally formed in glycolysis (Fig. 13-25). This constituted the chief stable primary product of photosynthesis by Calvin and Benson (1948).
When the period of photosynthesis was prolonged to 30 and then to 60 seconds, more and more compounds contained 14C atoms. They were triose and hexose phosphates.
It was concluded that phosphoglyceric acid was converted to carbohydrates by a reverse sequence of glycolytic reactions occurring in respiration via 3- phosphoglyceratdehyde (PGAL), fructose 1-6bisphosphate, glucose 6-phosphate and glucose 1-phosphate.
From glucose 1-phosphate, both starch and sucrose can be synthesized directly. After slightly longer periods of photosynthesis, labelled carbon also appeared in C4, C5 and C7 compounds, of the hexose monophosphate shunt.
Exposure time (sec) to 14CO2 product in which 14C appeared:
The living organisms have only one primary carboxylation mechanism. There are other carboxylations reactions also but they are derived from the primary mechanism. It is through this primary mechanism that all organic carbon is derived from CO2. This is called Calvin cycle, the Reductive Pentose Phosphate (RPP) pathway, Benson-Calvin cycle, the photosynthetic Carbon (reduction) cycle.
Features of Calvin Cycle:
The Calvin cycle has four main features:
1. Carboxylation:
In this part of the mechanism CO2 is linked with the ribulose 1, 5-bisphospate (RuBP) to yield two molecules of 4-phospoglycerate (PGA). This reaction is catalysed by RuBP carboxylase which is a complex enzyme having high affinity for CO2.
2. Reduction:
In this step PGA is reduced at the expense of assimilatory power to give rise to triosephosphate.
3. Regeneration:
In this step five molecules of triosephosphate are rearranged to regenerate 3 molecules of the CO2-acceptor.
4. Autocatalysis:
For every 3 molecules of CO2 entering RPP pathway one triosephosphate molecule is produced. The latter can be converted to starch, sucrose, etc. It can also enter the regenerative phase of the pathway leading to an autocatalytic build-up of intermediates.
Calvin—Cycle (C3 Cycle):
The reaction of carbohydrate synthesis occurs in a cyclic sequence of carboxylation reduction, hexoses formation and regeneration (Fig. 13.26, 27, 26A).
1. Carboxylation:
Carboxylation mechanism in photosynthesis must meet four criteria:
(i) Able to regenerate its CO2-acceptor,
(ii) Must work autocatalytically i.e. it should produce more potential substrate than it utilizes,
(iii) Its carboxylation reaction should have a very favourable equilibrium position to offset low CO2 in the atmosphere, and
(iv) Its carboxylase should have high affinity for CO2 to allow effective function in low CO2 .
In this phase a 5-carbon compound called ribulose monophosphate (RuMP) constitutes the starting point for the fixation and reduction of CO2.
In the following equation the whole process is summarized:
As will be observed RuMP combines with ATP to produce ribulose 1, 5-bisphosphate and the reaction is mediated by phosphoribulokinase. Ribulose 1, 5-bisphosphate then combines with one molecule each of water and CO2 and carboxydismutase enzyme affects this reaction.
Ribulosebisphosphate carboxylase (RuBP carboxylase) is the main enzyme of this reaction. It is the copper-containing enzyme which requires Mg2+ for its activity. In the end two molecules of 3-phosphoglyceric acid (PGA), 3-C compounds are produced.
It is presumed that RuBP changes into its enediol isomer and carbon is added at carbon-2 position to yield β-oxido-acid intermediate. Subsequently in the presence of water this intermediate splits into two molecules of 3-phosphoglyceric acid.
The latter is an important intermediate compound in plant metabolism. The compound can be identified even after 5 seconds of photosynthesis and can enter anabolic or catabolic chains.
The overall process is as follows:
CO2+ RuMP + 2NADPH2 + ATP” 2GAP + 2NADP + 3ADP + 2H3PO4
A part of glyceraldehyde 3-phosphate is transformed into its isomer dihydroxy acetone 3- phosphate with the help of phosphotrioseisomerase.
2. Hexoses Formation:
In this step in the presence of aldolase, one molecule each of dihydroxyacetone 3-phosphate and glyceraldehyde 3-phosphate condense together to form fructose 1, 6-bisphosphate.
Then one phosphate radical is removed to produce fructose 6-phosphate as follows:
Fructose 6-phosphate gives rise to fructose 1-phosphate and from the latter glucose 1-phosphate is formed. Glucose-1-phosphate in turn may react with ATP or UTP to produce ADP glucose or UDP- glucose.
ADP-glucose and UDP-glucose provide glucose residues which condense to form starch. This is the chief storage product in different plant cells and organs. It may be stated that sucrose is the first stable sugar produced during photosynthesis. UDP-glucose and fructose 6-phosphate condense in the presence of a phosphate to yield sucrose.
3. Regeneration of Ribulose 5-Phosphate:
In this step fructose 6-phosphate reacts with glyceraldehyde 3-phosphate to give rise to one molecule each of erythrose 4-phosphate and xylulose 5-phosphate with help of transketolsae.
Also erythrose 4-phosphate and dihydroxyacetone 3-phosphate molecules condense together in the presence of aldolase to produce sedoheptulose 1, 7diphosphate.Enzyme phosphatase in the presence of water converts sedoheptulose 1, 7 diphosphate into sedoheptulose 7-phosphate. In this step there is removal of phosphate radical.
There is also formation of one molecule each of xylulose 5-phosphate and ribose 5-phosphate by the combination of sedoheptulose 7-phosphate and flyceraldehyde 3-phosphate, in the presence of transketolase.
These 5-carbon compounds are transformed into ribulose 5-phosphate as shown below:
The essentials of Calvin cycle are shown below:
6 CO2 + 18ATP+ 12 NADPH ” F– 6−P+ 18 ADP+ 17 Pi+ 12NADP
Enzymes of Calvin Cycle:
In alfalfa when the amount of CO2 is increased, its rate of fixation is doubled but the pool sizes of Calvin cycle intermediates increase only marginally. Much attention is demanded for the RuBP carboxylase which in higher plants is composed of eight large and small subunits; of these the large subunits have three almost identical polypeptides whereas the small subunits have one or more types of polypeptides chain.
However, in some blue green algae, RuBP carboxylase lacks small subunits. In Anabaena cylindrica polyhedral bodies are shown to contain this enzyme while for higher plants it is postulated to be situated in the crystalline bodies in tobacco chloroplast stroma and even thylakoid of chloroplasts from ‘stressed’ spinach leaves. Apparently about 40% of leaf protein is RuBP carboxylase.
Some workers have proposed that this enzyme acts as nitrogen reserve available for the degradation and redistribution from older to younger parts of the plant. Now it has been conclusively shown that it has adequate affinity for subtrates and ample catalytic activity to support photosynthesis when activated by CO2 or Mg++. In the absence of these substances the enzyme reverts to the inactive state.
Available evidences tend to indicate that the enzyme undergoes conformational changes when subjected to temperature or Mg or RuBP, etc. It is on the large subunit that catalysis of RuBP carboxylase takes place. On the eight small subunits eight amino groups are present.
Each small subunit of the tobacco enzyme contains nearly seven lysyl residues. This enzyme also catalyses an oxygenase reaction giving rise to p-glycolate. The general observation is that carboxylase and oxygenase activities respond very similarly to effector molecules and CO2.
The precise nature of GAP dehydrogenase is still obscure and most of the available data point towards variable molecular weights. The spinach and Sinapsisalba enzymes are shown to have two subunits. Further in the presence of NAD+ and NADH” the enzyme tends to become sticky.
Glycerate-3-P kinase has also received very little attention and same is true of ribose-5-p isomerase. These two enzymes have been purified from leaves to homogeneity. Similarly reductive activation FBPase has been shown to be slow. When dissociated in two halves, this enzyme seems to require SBPase activity.
The protein factor needed for activation of FBPase and SBPase by reduced ferredoxin comprising two components which are thioredoxin and ferredoxin-thioredoxinreductase. Chloroplast malate dehydrogenase (NADP) was also activated in this system. There is a definite need to search for the alternative activating systems.
Some workers believe ditliiol-containing light effect mediators are associated with the photosynthetic electron transport system. Further glucose 6-phosphate dehydrogenase enzyme is inactivated by these light mediators following illumination.
In general enzymes activated in vivo by light mediated processes are deactivated in the dark and the precise nature of the deactivation is not clear. Possibly glutathione may be involved in this.
Role of light in the activation of ‘Dark phase’ enzymes:
It is generally implied that light was chiefly involved in the generation of ATP and NADPH required to drive Calvin cycle. In recent years it has come to light that at least five enzymes of Calvin cycle are activated by light and these are e.g., RuBP carboxylase, glyceraldehyde 3-phosphate dehydrogenase, fructose bisphospatase, etc.
It seems untenable to use the term ‘Dark phase’. Light affects several changes within the chloroplast stroma which in turn activate or deactivate the mentioned enzymes. Light has been shown to affect several processes including promotion of the reduction of compounds which are termed ‘light effect mediators’ and these in turn activate some of these enzymes.
Control Mechanisms:
Several control points have been proposed which regulate the Calvin cycle. In the first place, RPP pathway constitutes an efficient auto-catalytic device. Thus, for each turn of the cycle, several starter molecules (RuBP) are produced. Consequently, the rate of operation of the cycle can be enhanced by building its own intermediates.
It is essential since of the intermediates like triose phosphate, glycolate and PGA could leave the chloroplast during darkness, leaving the intermediates in low concentration. Apparently the level of intermediates must be boosted in order that cycle operates with rapidity. The cycle may also be limited by the regulatory reactions involving enzymes activation by light.
Clearly, in a leaf kept under darkness, carboxylases become inactive and same is true of phosphatases. Further, the balance of the synthesis of hexoses, pentoses, trioses, PGA, etc. is maintained through allosteric effects of various reactions in the cycle by cofactors like ATP or ADP, etc. In several ways, balance operations are possible and the cycle reacts with rapidity to the demands of other parts of the cell for several different products.
Energy Balance:
Considering the overall reactions of the cycle, it may be recalled that 6 CO2 combine with 18 ATP and 12 NADPH to yield one hexose (C6 H12 O6), 18 ADP+Pi, 12 NADP and 6 of H2 O. 18 ATP has a total of nearly 140 kcal whereas 12 NADPH a total of about 615 kcal. Taken together the energy input is nearly 755 kcal whereas in hexose about 670 kcal/mol of energy is recovered.
On per cent basis, the efficiency may be calculated as 90 while rest of the 10 per cent energy keeps the cycle going. This also indicates the efficiency of the cycle in storing the chemical potential harnessed from light energy. We have already pointed towards the positive feedback system of the Calvin-Benson cycle.