10 april 2008 dark reactions of photosynthesis andy howard introductory biochemistry 10 april 2008
TRANSCRIPT
10 April 2008 Dark Reactions p. 2 of 47
Dark reactions matter!
Not all of these reactions really take place in the dark; but some do, and even the ones that take place in daylight are not directly dependent on photon absorption
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What we’ll discuss Dark reactions of
Photosynthesis RuBisCO Calvin Cycle
overview C5 to C3 to C6 Regenerating C5’s Energy bookkeeping
Sucrose & Starch Other C-fixation
paths
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Dark reactions
Series of ordinary chemical reactions Powered by reducing power in NADPH Anabolic Some common features with pentose
phosphate pathway
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Dark reactions: overview
RuBisCO fixes atmospheric CO2 into carbon skeletons
Reductions of 3-phosphoglycerate build up carbohydrate
Pathway is cyclic in that RuBP is regenerated for additional reactions
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RuBisCO reaction Condensation of ribulose
1,5-bisphosphate (RuBP) with CO2 to produce two molecules of 3-phosphoglycerate
Enzyme is ribulose1,5-bisphosphate carboxylase / oxygenase(RuBisCO)
RuBP
3-phosphoglycerate
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The unwanted (?) side-reaction of RuBisCO
Secondary reaction isribulose 1,5-bisphosphate+ O2 3-phosphoglycerate +2-phosphoglycolate
Uses up oxygen rather than CO2
No net carbon incorporation into organic molecules
2-phospho-glycolate
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RuBisCO structure L8S8 stoichiometry
in higher plants:Mol.Wt. L=55kDa;Mol. Wt. S=12 kDa
TIM barrels in both All (?) catalytic activity in L
(large) subunit L coded for by chloroplast gene S by nuclear genome Does S play a controlling role?
PDB 1WDDOctamer of L8S8 unitsL2S2 shownfrom rice(cf. fig. 15.21)
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RuBisCO regulation Plant growth closely associated with
carboxylation / oxygenation ratio:Carboxylation high means fast growth
Easy way to alter that: grow plants in high CO2
Difficult to do that without animal toxicity! Expensive to put your cornfield in a plastic
bubble (but not impossible)
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Could you win genetically? Attempts to engineer proteins that
don’t do oxygenation(or even that have improved CO2/O2 activity ratios) have failed
There are some plants whose RuBisCO has a better SC/O than that of others
Maybe O2 and CO2 bind in precisely the same way!
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Subsequent dark reactions, I Pair of 3-phosphoglycerate
molecules enter reductive pathway toward bigger sugars
Note that this reaction appears in glycolysis (in reverse) and in gluconeogenesis
Phosphoglycerate kinase activation:3-P-glycerate + ATP 1,3-bisP-glycerate + ADP PDB 1PHP
43 kDa monomerBacillus stearothermophilus(unfortunately!)
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Subsequent dark reactions, II (cf. fig. 15.18)
Three glycolysis / gluconeogenesis rxns: GAPDH reaction:
1,3-bisP-glycerate + NADPH + H+glyceraldehyde-3-phosphate + NADP + Pi
TIM required to convert G3P to DHAP Aldolase makes fructose 1,6-bisphosphate
Some RuBP is recycled back in to provide input to subsequent condensations with CO2
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RuBisCO, revisited 2-phosphoglycolate is the product
of the oxygenation reaction 2-P-glycolate is decarboxylated:
2 2-P-glycolate CO2 + 3-P-glycerate +Pi
The 3-P-glycerate can re-enter the Calvin cycle, but at the cost of some carbon
This lossy pathway is known as photorespiration
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Be careful how you describe transketolase and transaldolase
A few days ago we said (in lecture) that the transketolase reaction wasKn + Am Kn-2 + Am+2
That’s wrong: we do donate two carbons from the ketose to the aldose, but they swap carbonyl positions when you do, so the reaction is really Kn + Am An-2 + Km+2
The notes have already been corrected!
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Calvin cycle: first reaction
Begins with ATP-dependent phosphorylation of 3-phosphoglycerate to make 1,3-bisphosphoglycerate via phophosphoglycerate kinase
Same reaction found in gluconeogenesis; reverse of glycolytic step
Enzyme is 3-layer sandwich
PDB 1V6S86 kDa dimerThermus thermophilusMonomer shown
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2nd Calvin-cyclereaction: GAPDH
NADPH-dependent reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate
As in gluconeogenesis, reverse of glycolytic reaction
GAPDH: typical NAD(P) dependent oxidoreductase
PDB 1RM4297 kDa octamerdimer + monomer shownspinach
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The fates of glyceraldehyde-3-phosphate The pathway divides three ways at this
metabolite One equivalent toward fructose 1,6-
bisphosphate and gluconeogenesis Two head toward pentose phosphate
pathway, where a second bifurcation happens
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C3 to C6 TIM converts one molecule of
glyceraldehyde 3-phosphate to dihydroxyacetone phosphate
Glyc-3-P and DHAP condense to form fructose 1,6-bisphosphate in standard aldolase reaction
Fructose 1,6-bisphosphatase removes the 1-phosphate to make fructose 6-phosphate
All of this happens in gluconeogenesis
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Transketolase As we saw in the PPP,
fructose-6-P can react with glyceraldehyde-3-P in a transketolase reaction to form xylulose-5-phosphate and erythrose-4-phosphate
K6 + A3 A4 + K5 Typical TPP binding
structure
PDB 1ITZ297 kDa octamerdimer+monomer shownmaize
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Fates of DHAP Can participate in F-6-P production Can condense with erythrose-4-P in an
aldolase reaction to form sedoheptulose 1,7-bisphosphate (K3 + A4 K7)
This can be dephosphorylated at the 1-position to form sedoheptulose 7-P via sedoheptulose 1,7-bisphosphatase
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The final Glyc3-P
It can condense with sedoheptulose 7-phosphate in another transketolase reaction to form xylulose-5-phosphate and ribose-5-phosphate:K7 + A3 A5 + K5 (fig. 15.19)
The ribose-5-phosphate is an endpoint but it can also be isomerized to ribulose-5-phosphate
Xylulose-5-phosphate can be epimerized to form ribulose-5-phosphate too
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Activation ofribulose-5-phosphate
Phosphoribulokinase uses ATP as a phosphate source to convert ribulose-5-phosphate to RuBP
Enzyme is similar to adenylate kinase
PDB 1A7J32 kDa monomerRhodobacter sphaeroides
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What is unique here?
Not much Last reaction is specific to Calvin cycle Others are found in gluconeogenesis or
pentose phosphate pathway or both In this direction these reactions require
the NADPH and ATP derived from the light reactions of photosynthesis
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Bookkeeping for dark reactions
Numbers given on fig.15.19 presuppose 3 input RuBP molecules per run of the cycle
This makes it easy to divide up the Glyceraldehyde 3-P later
Net reaction is:3 CO2 + 9ATP + 6 NADPH + 5 H2O glyceraldehyde 3-P + 9ADP +8 Pi + 6 NADP+
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Cost of making Acetyl CoA• We get back 2 NADH, 2 ATP when we
convert glyceraldehyde 3-P to acetyl CoA• Therefore acetyl CoA costs 9-2 = 7 ATP
and 6-2=4 NAD(P)H• At 2.5 ATP per NAD, that total is 7 + 2.5 *
4 = 17 ATP required per acetyl CoA• When we oxidize acetyl CoA we get 10
ATP (see TCA-cycle lecture),so we’re 10/17 = 59% efficient
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Carbohydrate storage in plants Glyc3P is converted to glucose-6-P or
glucose by gluconeogenesis Glycogen is storage polysaccharide in
bacteria, algae, some plants Other plants make starch (amylose or
amylopectin) from glucose-6-P Pathway begins with conversion of
glucose-6-P to glucose-1-P, catalyzed by phosphoglucomutase
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Starch synthesis Glucose 1-P activated with ATP, not UDP
-D-glucose 1-P + ATP ADP-glucose + PPi
Reaction driven to the right by hydrolysis of PP i
ADP glucose is added to growing starch molecule with release of ADP:ADP-glucose + (Starch)n ADP + (Starch)n+1
Branching in amylopectin accomplished as in glycogen(Yao et al (2004) Plant Physiol. 136:3515)
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Diurnal variations in starch
Starch synthesis in daylight:ATP is readily available
Starch degradation at night Starch phosphorylase cleaves
starch to produce glucose-1-phosphate;glucose-1-P to triose phosphates by glycolysis
Enzyme is similar to glycogen phosphorylase
PLP-dependent
PDB 2C4M350 kDa tetramerCorynebacterium callunae
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Alternative path for night-time starch degradation
Starch to dextrins via amylase Dextrins are oligosaccharides
beginning with a -1,6 link Dextrins eventually degraded
to glucose Glucose is phosphorylated by
hexokinase Enzyme:
sheet domain + TIM barrel
PDB 1HT645 kDa monomerbarley
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Sucrose: mobile carbohydrate
Synthesized in chloroplast-containing cells; exported tovascular system so otherplant parts can use it
Two fructose 6-phosphatemolecules are starting points(fig 15.25)
One is converted to Glucose-1-P (via glucose 6-P) and thence to UDP-glucose
That condenses with the other Fructose-6-P with the help of sucrose 6-P synthase to form sucrose 6-P
That gets dephosphorylated to make sucrose
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Enzymes in sucrose synthesisEnzyme Reactant Product Glucose 6-phosphate
isomerase F-6-P G-6-P Phosphoglucomutase G-6-P G-1-P UDP-glucose G-1-P + UDP-
glucosepyrophosphorylase UTP + PPi
Sucrose 6-phosphate F-6-P + Sucrose-6-Psynthase UDP-glucose
Sucrose phosphate Suc-6-P Sucrosephosphatase + H2O + Pi
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UDP-glucose pyrophosphorylase
Catalyzesglucose-1-P + UTP UDP-glucose + PPi PDB 2ICY
103 kDa dimerArabidopsis
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Sucrose 6-phosphate phosphatase
Contains “tongs” that release free sucrose into the cell:Fieulaine et al, Plant Cell 17: 2049-2058
Rossmann fold + complex
PDB 1TJ527 kDa monomerSynechocystis
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How sucrose is used Sucrose taken up by non-photosynthetic
cells Broken down to glucose and fructose
supplies energy by glycolysis and TCA Glucose and fructose can be built back up to
starch in storage tissues:Amyloplasts (modified chloroplasts with no photosynthetic mechanisms) in root cells do this
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Other carbon-fixation pathways Purpose: increase local [CO2] / [O2] to
improve performance of RuBisCO C4 pathway (high temp, lots of light) Crassulacean acid metabolism (high
temp, limited water)
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C4 pathways Common in maize, sorghum, sugarcane,
weeds Needed at high temp because
rate(oxidation)/rate(carboxylation) increases with temperature
External CO2 acceptor is PEP via PEP carboxylase; product is oxaloacetate
This occurs in mesophylls; bundle sheath cells continue to do ordinary RuBisCO-based carbon fixation using CO2 released from metabolites
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PEP Carboxylase
PEP + HCO3-
oxaloacetate + Pi
Occurs outside C4 metabolism too
One TIM barrel per monomer
PDB 1JQO427 kDa tetramermaize
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Crassulacean acid metabolism
Leaf cells open to CO2 uptake lose a lot of water during the day(high evaporation rate)
Solution: assimilate carbon at night Reactions are as in C4 pathway;
cellular specialization and enzyme regulation are different
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Stomata and vacuoles Stomata (spaces between cells that
can open to allow access for respiration) near mesophylls open only at night, enabling PEP carboxylation to oxalacetate and then reduction to malate
Malate stored in central vacuole, then released during the day when the stomata are closed
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CAM: day and night
University of Newcastle, Plant Physiology program
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iClicker quiz question 1 Oxidation of a 2n-
carbon fatty acid yields (n-1) QH2,(n-1) NADH, and n acetyl CoA. Initiating the process costs 2 ATPs. Assume we can get 10 ATP per acetyl CoA. How much ATP can we get from oxidizing palmitate?
(a) 104 ATP (b) 106 ATP (c ) 108 ATP (d) 112 ATP (e) Undeterminable
given the data supplied
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Answer to 1st question Palmitate is a C16 carboxylic acid.
Therefore in the conditions of the problem, 2n = 16, n = 8, n-1 = 7.
Thus we get 7 QH2, 7 NADH,8 acetyl CoA produced by its oxidation
Thus we get 7*2.5 + 7 * 1.5 + 8 * 10 = 17.5 + 10.5 + 80 = 108 ATP produced
Starting the process costs 2 ATP, so the net result is 106 ATP gained
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iClicker quiz question 2Why would you not expect to find crassulacean
acid metabolism in tropical plants? (a) Tropical plants do not photosynthesize. (b) Tropical plants cannot develop the stomata
that close off the chloroplast-containing cavities (c) Water conservation is less critical in areas of
high rainfall (d) The waxy coating required to close off the
leaves’ access to O2 would dissolve in the high humidity and high temperature of the tropics
(e) None of the above
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Answer: (c)
The primary significance of CAM is conservation of water in regions of low humidity, where evaporation rates are high and water is scarce. Neither of these conditions pertains in the tropics.
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Control of CAM PEP carboxylase inhibited by
malate and low pH That prevents activity during
daylight, which would lead to futile cycling and competition for CO2 between PEP carboxylase and RuBisCO