Unicellular Photosynthetic Pathways
Regulation of Carbon Metabolism
1)Substrate level regulation
2)pH level regulation
Activase and Inhibitor mediated regulation of Rubisco activity
2)Equilibrium kinetics and Rubisco activation
a)CA1P and transition state analogues
Carbon fixation uses ATP and NADPH generated from the light reactions of photosynthesis to "fix" CO2 into the hexose sugar, glucose. The overall reaction of CO2 fixation is:
The acceptor for CO2 fixation was found to be the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). The enzyme ribulose bisphosphate carboxylase/oxygenase or RUBISCO carries out this reaction.
The RUBISCO catalyzed CO2 fixation reaction:
Enzymatic abstraction of the C-3 proton of RuBP yields a 2,3-enediol intermediate (I), which is stereo-specifically carboxylated at C2 to create the 6C ß-keto acid intermediate (II), 3-keto-arabinitol. This is rapidly hydrated to form the gem-diol (III). Deprotonation of the C3 hydroxyl and cleavage results in two 3-phosphoglycerates (3-PG). Mg+2 at the active site of RUBISCO aids in stabilizing the 2,3-enediol transition state for CO2 addition and facilitates the carbon-carbon bond cleavage that leads to two 3-C products. CO2, not HCO3- (its hydrated form) is the actual substrate.
The set of reactions that transform 3-PG into hexose is known as the Calvin or Calvin-Benson cycle. This reaction is indeed cyclic since not only is carbohydrate generated as an end product, but also the 5C acceptor, RuBP, is regenerated. P>
Fifteen enzymes catalyze 15 reaction steps of the Calvin-Benson Cycle:
Reactions 1 through 15 constitute the cycle that culminates in the formation of one equivalent of glucose. The enzyme catalyzing each step, a concise reaction, and the overall carbon balance is given. Numbers in parentheses denote the numbers of carbon atoms in the substrate and product molecules. Prefix numbers indicate in a stoichiometric fashion how may times each step is carried out in order to provide a balance net reaction.
|1||Ribulose bisphosphate carboxylase: 6 CO2 + 6H20 + 6 RuBP ® 12 3-PG||6(1) + 6(5) ® 12(3)|
|2||3-Phosphoglycerate kinase: 12 3-PG + 12 ATP ® 12 1,3-BPG + 12 ADP||12(3) ® 12(3)|
12 1,3-BPG + 12 NADPH ® 12 NADP+ + 12G3P + 12 Pi
|12(3) ® 12(3)|
|4||Triose-P isomerase: 5 G3P® 5 DHAP||5(3) ® 5(3)|
|5||Aldolase: 3 G3P + 3 DHAP ® 3 FBP||3(3) + 3(3) ® 3(6)|
|6||Fructose bisphosphatase: 3 FBP + 3 H20 ® 3 F6P + 3 Pi||3(6) ® 3(6)|
|7||Phosphoglucoisomerase: 1 F6P ® 1 G6P||1(6) ® 1(6)|
|8||Glucose phosphatase: 1 G6P + 1
H20 ® 1 GLUCOSE + 1
The remainder of the pathway involves regenerating six RuPB acceptors (=30C) from the leftover two F6P (12C), four G3P (12C), and two DHAP (6C).
|1(6) ® 1(6)|
|9||Transketolase: 2 F6P + 2 G3P ® 2 Xu5P + 2 E4P||2(6) + 2(3) ® 2(5) + 2(4)|
|10||Aldolase: 2 E4P + 2 DHAP ® 2 sedoheptulose-1,7-bisphosphate (SBP)||2(4) + 2(3) ® 2(7)|
|11||Sedoheptulose bisphosphatase: 2 SBP + 2 H20 ® 2 S7P + 2 Pi||2(7) ® 2(7)|
|12||Transketolase: 2 S7P + 2 G3P ® 2 Xu5P + 2 R5P||2(7) + 2(3) ® 4(5)|
|13||Phosphopentose epimerase: 4 Xu5P ® 4 Ru5P||4(5) ® 4(5)|
|14||Phosphopentose isomerase: 2 R5P ® 2 Ru5P||2(5) ® 4(5)|
|15||Phosphoribulose kinase: 6 Ru5P + 6 ATP ® 6 RuBP + 6 ADP||6(5) ® 6 (5)|
|Net: 6 CO2 + 18 ATP + 12 NADPH + 12H+ + 12 H20 ® glucose + 18 ADP + 18 Pi + 12 NADP+||6(1) ® 1(6)|
The 2 reactions of RUBISCO can be simply put as follows:
The relative rates of these 2 reactions are determined by the affinity of the enzyme for CO2 and O2 (Km), the Vmax values for the carboxylase and oxygenase activities, the concentrations of CO2 and O2 and temperature. Carboxylase activity is competitively inhibited by O2 and the inhibitor constant (Ki) for O2 is similar to the Km value of the oxygenase activity for O2. Likewise, the oxygenase activity is competitively inhibited by CO2 and the Ki for CO2 is ~ the Km value of carboxylase activity for CO2.
RUBISCO has much greater affinity for CO2 (Km ~10 µM) than for O2 (Km ~535 µM) making the ratio of the 2 activities much more sensitive to changes in CO2 than to O2 concentrations. The concentration of CO2 dissolved in H2O at 20°C is also ~10 µM, but the level in a mesophyll of a leaf in air is ~5 µM. The O2 concentration in a leaf at 20 °C is ~270 µM. Under these conditions RUBISCO catalyzes both carboxylation and oxygenation of RuBP. Under normal atmospheric conditions most C3 plants metabolize ~70% of RuBP via the carboxylase activity and ~30% by the oxygenase activity of RUBISCO.
This value has apparently improved during evolution varying from ~10 in bacteria to ~50 in cyanobacteria and about 80 in higher plants, but has not been eliminated during evolution (nor have any researchers yet been able to eliminate the oxygenase activity of RUBISCO). Oxygenation occurs by direct attack of O2 on the 2,3-enediol intermediate and is hypothesized to be an inevitable consequence of the reaction mechanism of RUBISCO.
The principal factor influencing the relative rates of carboxylation and oxygenation, apart from CO2 and O2 concentrations, is temperature. High temperatures promote oxygenation, and hence photorespiration, 2 ways. 1st, the solubility of CO2 in water decreases more rapidly than that of O2 as the temperature increases. For example, at 10°C the O2/CO2 solubility in water is 20, yet at 40°C it is 28. 2nd, the specificity factor, , of RUBISCO declines with increasing temperature from 7°C to 35°C. This happens since the reaction of the 2,3-enediol intermediate (see above) with O2 has a higher free energy of activation than the reaction with CO2. Thus oxygenation is more sensitive to temperature than carboxylation and increases more rapidly as the temperature rises.
The phosphoglycolate (glycolate-2-P) produced by the oxygenation of RuBP by RUBISCO cannot be directly utilized by the Calvin cycle. Instead, it is salvaged in the photorespiratory pathway with loss of energy and one of the fixed carbons.
The main functions of this pathway are the conversion of the 2C molecule, glycolate-3-P to glycine and decarboxylation of 2 molecules of glycine to serine, CO2 and NH3. The 3C molecule, serine, is then converted into G-3-P which re-enters the Calvin-Benson pathway. One fourth of the carbon in glycolate is released as CO2 (hence the name photorespiration). The NH3 is refixed by glutamine synthetase and glutamine synthase. The CO2 released can be refixed by the carboxylation reaction of RUBISCO, but the energy expended in photorespiration is still lost.Photorespiration represents a very large energy expenditure by plants. It requires as much NADPH input as the carbon reduction cycle and 13% more ATP:
|All materials © 1998, 1999, 2000, Dr. David Hildebrand or Dr. Bob Houtz, unless otherwise noted.|
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