Once 3-PGA is formed the reactions of glycolysis/gluconeogenesis interconvert it to Ga-3-P and F-6-P, intermediates of the pentose-P pathway, and DHAP. These intermediates are then interconverted to reform Ru-5-P. For every three carbon dioxides incorporated by RuBisCo, one extra 3-PGA is formed which can be used for the synthesis of glucose etc. The overall stoichiometry of the cycle is shown on the Calvin Cycle Flow Diagram. This diagram emphasizes recycling vs. incorporation. (packet)
Calvin Cycle regulation: Ribulose-1,5-bis phosphate carboxylase is the flux generating enzyme of the Calvin cycle, and is the main regulatory enzyme of this cycle. Velocity vs [S] plots yield hyperbolic kinetics vs. ribulose-1,5-bis P, but give sigmoidal kinetics vs. carbon dioxide. The activity of the enzyme increases with increasing pH between pH 7 & 9 (H+ is a (-) effector; recall that with illumination the stromal pH increases due to proton pumping by the light reactions). NADPH and Mg2+ are both positive effectors, both increasing with illumination. A switching mechanism turns RuBisCo off in the dark and back on in the presence of light.
FYI - Additional Regulation
A number of additional enzymes of the cycle are regulated in addition to RuBisCo. These enzymes show differences in activity with illumination. In this case the light effects the enzyme via a reduction path leading from the light reactions which provide electrons to reduce a disulfide on thioredoxin, which can in turn reduce disulfides on the enzymes which, for example, increases the rate for F-1,6-bis phosphatase, and changes the substrate specificity for the Ga-3P DH form NADH in the dark to NADPH in the light. Thus the system favors glucose synthesis in the light and glycolysis in the dark!
Background on plant strategies under high heat/light low water conditions
C4 Plants: These plants thrive in environments with high temperatures and low humidities where the stomata in the leaves must be closed. Under these circumstances carbon dioxide concentrations fall in the leaves while oxygen rises, favoring photorespiration over photosynthesis and greatly reducing productivity. (In photorespiration oxygen binds competitively with carbon dioxide at the active site of RuBisCo: the net result is that Ru-1,5-bis P is oxidized and energy and carbon are lost instead of gained: Ru-1,5-bis P 3-PGA + 3-P-Glycolate, 2 P-Glycolate 3-PGA + CO2.) In C4 plants the photosynthesizing cells are protected from the atmosphere by a layer of mesophyll cells. In these cells the PEP carboxylase reaction is used to capture carbon dioxide, with the resulting oxaloacetate carbons transported to the photosynthesizing cell. The first compound incorporating the carbon dioxide thus has four carbons and hence the name (unlike in the Calvin cycle where the first labeled compound, PGA is C3). The carbon dioxide is then released and used in the Calvin cycle. Note that these plants are investing extra energy from ATP to concentrate carbon dioxide. However, they tend to live in high light environments where cyclic Photophosphorylation can be used to make up this extra ATP with little trouble. A variety of transport mechanisms exist in different plant groups. In another mechanism, CAM (Crassulacean), carbon dioxide is taken up at night and incorporated into malate. The malate is then used the next day to make PEP. Thus the plants can keep their stomata closed during the day].
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Protein catabolism and anabolism are often out of sync - either no additional protein is needed or the amino acid composition of the synthesized proteins is not identical to the protein being hydrolyzed. Neither protein nor amino acids are stored as such. Thus organisms must frequently degrade excess amino acids. Two paths may be available: deaminate the unneeded amino acids and breakdown the carbon skeletons for energy or for storage as fat or carbohydrate and eliminate the nitrogen; or the nitrogen may be transferred to another carbon backbone to make a needed amino acid.
Nitrogen can be eliminated in a variety of forms, depending on the physiological conditions experienced by the organism:
Amino Acid Transamination & Deamination
For any of these products nitrogen must first be removed from the amino acids. Ammonia is often an intermediate if not the final product in all of these organisms. Most ammonia results from the deamination of a single amino acid: glutamate, via the Glutamate dehydrogenase reaction:
This enzyme is somewhat unusual in that it can use either NADH or NADPH. Glutamate DH is activated by ADP and inhibited by GTP in vitro so they may regulate it in vivo. Of course these nucleotides would not provide a highly sensitive regulation, activity would only loosely follow energy charge.
How are the other amino acids deaminated? Most are transaminated, transferring their N to make glutamate:
amino acid + 2-oxoglutarate 2-oxoacid + glutamate
There are three main transaminases or Amino transferases in liver, all requiring Pyridoxal-P as a cofactor:
The various aminotransferases in the liver all funnel excess N to glutamate and aspartate. Glutamate can then be deaminated by Glutamate DH to give ammonia, contributing up to 1/2 of the N in urea. Aspartate provides N directly to urea synthesis, contributing 1/2 of urea N.
Let's look at the aminotransferase reaction and the cofactor pyridoxal-phosphate. Pyridoxal-P (PLP) is derived from vitamin B6 (pyridoxine) via phosphorylation. Vitamin B6 is thus essential for protein metabolism.
Pyridoxal-P is used to form Schiff bases with its various substrates, but in this case the nitrogen is provided by the substrate. PLP acts as an electrophilic catalyst; since none of the amino acids are electrophilic, cofactors must be provided when electrophilic covalent catalysis is needed.
As we saw before the transaminases catalyze symmetrical reactions:
An amino acid reacts with a keto acid to give an amino acid and a keto acid. Thus one might expect a symmetrical mechanism, which is in fact the case. In the diagram below only the first half of the reaction mechanism is shown. The second half simply "reflects" the first half. It involves substituting the second keto acid in the right-hand step and reversing the process to take the second amino acid off in the left-hand step to complete the reaction (change the R s to R' s in the reverse steps):
Note that the PLP starts out covalently linked to a lysine-N, which is displaced by the reactant amino acid. During the remainder of the reaction the PLP is held non-covalently in the active site. In the final step the lysine amino group on the enzyme displaces the product amino acid nitrogen to release the free amino acid.
Chemically the reactant amino acid is oxidized (aldimine-ketimine shift in the mechanism) to release the keto acid product. The keto acid reactant is then reduced (ketimine-aldimine shift) to release the amino acid product. As might be expected the enzyme exhibits Ping-Pong Bi Bi kinetics:
© R. A. Paselk 2010;
Last modified 26 April 2013