20: Carbohydrate biosynthesis (plants and bacteria)

Question 1

what are dark reactions?

Answer 1

carbon assimilation reactions. the reactions that use ATP and NADPH to make carbohydrates from CO2. called dark because they were thought to have occurred when plants were in the dark, but this isn’t really accurate.

Question 2

what is a carbohydrate?

Answer 2

chemically defined as polyhydroxy aldehyde or polyhydroxy ketones. complex substances which on hydrolysis yield polyhydroxy aldehyde/ketone.
(CH2O)n is general formula for most sugars
carbons must by more than 3. 3 = triose

Question 3

where are trioses made?

Answer 3

during the Calvin cycle in the chloroplast stroma

Question 4

stages of the Calvin cycle. draw it

Answer 4

1. carbon fixation
2. Glyceraldehyde 3-phosphate generation
3. regeneration of ribulose 1,5
   Pt 1 slide 7 drawing

Question 5

describe step 1 of Calvin cycle

Answer 5

Fixation of CO2 by rubisco
reactants: CO2, ribulose 1,5 bisphosphate
products: 2 3-phosphoglycerates
enzyme: rubisco
very slow, low turnover enzyme. plants get around this by having a bucketload, as much as 50% of total soluble protein. rubisco must be activated via carbamoylation

Question 6

mechanisms of step 1 of Calvin cycle

Answer 6

Rubisco mechanism
pt 1, slide 9
Mg2+ is critical. must be activated via carbamoylation. CO2 from environment is present on 1/2 of molecules produced.

Question 7

how is rubisco regulated?

Answer 7

post translational modification: when the Lys is not carbamoylated a ribulose 1,5-bisphosphate binds tightly and the molecule is inactive. ATP dependent removal of the ribulose allows for the Lys to be carbamoylated, Mg to associate, and rubisco to be active.
2-carboxyarabinitol 1-phosphate: a naturally occurring molecule produced in the dark. It is similar to the b-ketoacid hydrated intermediate and so binds rubisco, blocking activity when dark.
pt 1, slide 11

Question 8

describe step 2 of Calvin cycle

Answer 8

Formation of glyceraldehyde 3-phosphate
reactants: 3-phosphoglycerate, ATP, NADPH
products: glyceraldehyde 3-phosphate, ADP, NAD+, Pi
enzyme: not in slides…
important to count carbons, this reduction uses 6 3pg’s and makes 6 g3p’s. It costs 6 ATP and 6 NADPH. This amount of carbons is required in order to allow 3 carbons to leave the cycle and 15 carbons to stay as ribulose

Question 9

mechanism of step 2 Calvin cycle

Answer 9

formation of glyceraldehyde 3-phosphate
pt 1, slide 12
costs ATP and NADPH. goes through 1,3-bisphosphoglycerate intermediate. same as glycolysis steps backwards

Question 10

describe step 3 of Calvin cycle

Answer 10

regeneration of ribulose 1,5-bisphosphate
reactants: 5 glyceraldehyde-3-phosphate
products: 3 ribulose 1,5-bisphosphate
9 step process with important intermediates fructose-1,6-bisphosphate and fructose-6-phosphate. Interconverts between 3 and 7 C molecules. Pi is lost during the conversion of ribulose 5-phosphate to ribulose 1,5 bisphosphate, this causes an issue.

Question 11

Review the stoichiometry of the Calvin cycle

Answer 11

In order for one 3-carbon molecule (glyceraldehyde-3-phosphate) to leave the cycle, 6 molecules must be made. 5 of them stay in the cycle and reform the 3 ribulose 5-phosphate molecules. other molecules are needed in this order: 3 CO2, 6 ATP, 6 NADPH and 6 H+, 3 ATP. Total of 9 ATP and 6 NADH, a 3:2 ratio!
see pt 1 slide 12 and 15.

Question 12

explain the need for the Pi/triose phosphate antiporter

Answer 12

Pi is lost during the Calvin cycle during the final step of regeneration as it is incorporated into ribulose 5-phosphate. To replenish the Pi, an anitporter moves Pi into the stroma and moves dihydroxyacetone phosphate (made from glyceraldehyde-3-phosphate from Calvin cycle) out into the cytosol.

Question 13

Write the net reaction for carbon fixation reactions in plants (from CO2 to sugar)

Answer 13

6 CO2 + 18 ATP + 12 NADPH + 12 H+ + 12 H2O –> glucose + 18 ADP + 18 Pi + 12 NADP+

Question 14

what happens in the stroma (Calvin cycle) with illumination?

Answer 14

The light reactions increase activity. NADPH and ATP conc. increase, H+ transfer out of stroma increases (increases pH of stroma), Mg2+ flows into stroma.

Question 15

how does light indirectly regulate Calvin cycle enzymes?

Answer 15

The conditions that result from active light reactions (high NADPH, ATP, pH, and Mg2+ in stroma) cause rubisco activation and fructose 1,6-bisphosphatase activation. Another regulation occurs in enzymes with cysteine residues that are susceptible to oxidation. In the dark, oxidation occurs and a disulfide bond forms, inactivating the enzyme. In the light, some light energy from PSI can be diverted to thioredoxin which will correct the enzyme oxidation, causing it to be active. Basically the cycle runs faster in the light.

Question 16

why does rubisco have issues?

Answer 16

rubisco is naughty and promiscuous. it lacks specificity and will produce 2-phosphoglycolate instead of 3-phosphoglycerate when it uses O2 instead of CO2. this is a very costly side reaction of the carbon assimilation reactions as it ties up 2 carbons and releases CO2 while being corrected

Question 17

how is the naughty rubisco problem corrected?

Answer 17

the glycolate salvage pathway. This pathway takes two 2-phosphoglycolates and creates a serine and CO2. It takes place in 3 compartments: chloroplast, peroxisome, and mitochondrion. It’s a long and obnoxious pathway, smart plants try to avoid using it. Costs ATP and NADPH, consumes O2 and produces CO2
pt 2 slide 4

Question 18

how do C4 plants work?

Answer 18

CO2 fixation and rubisco activity are spatially separated. CO2 is temporarily fixated into a 4C unit (oxaloacetate) in the mesophyll cell while the rubisco is located in bundle sheath cell. Malate and pyruvate are transported between the two spaces. This is more costly and requires more ATP

Question 19

how do CAM plants work?

Answer 19

CO2 fixation and rubisco activity are temporally separated. The stomata reduces loss of water through pores, stomata allows night entry of CO2 and is closed during day. CO2 is fixed into oxaloacetate (4C), reduced to malate, and stored in vacuoles at night. In the day the malate releases and CO2 is delivered to rubisco

Question 20

what type of plants are C4 and CAM? where do they live?

Answer 20

C4: tropic plants. lots of energy to spare so can afford more costly CO2 fixation
CAM: desert succulents. evolved to reduce water loss during the day

Question 21

quick compare and contrast the types of plant photorespiration

Answer 21

C3: Calvin cycle in mesophyll, uses glycolate cycle to correct for naughty rubisco
C4: spatially separated in mesophyll and bundle sheath to prevent naughty rubisco
CAM: temporally separated CO2 fixation at night and Calvin cycle during day to prevent naughty rubisco

Question 22

what is the substrate for starch biosynthesis?

Answer 22

ADP-glucose. ADP is required bc it is a good leaving group and makes the process thermodynamically favorable

Question 23

biosynthesis of starch mechanism

Answer 23

pt 2 slide 12
starch synthase has 2 nucleophilic residues. the extending sugar chain see-saws back and forth between the two residues until a molecular ruler limits the size

Question 24

what is the substrate for sucrose biosynthesis?

Answer 24

UDP-glucose. UDP is good leaving group

Question 25

biosynthesis of sucrose mechanism

Answer 25

pt 2, slide 14-15
fructose 6-phosphate is made during step 3 of Calvin cycle. This is joined with UDP-glucose via sucrose 6-phosphate synthase and phosphatase.

Question 26

what are the three products that triose phosphates are important for? what does this mean in terms of regulation?

Answer 26

triose phosphates can be used in the Calvin cycle (making ribulose 1,5-bisphosphate), to make sucrose, and to make starch. Balance between the three must be regulated and coordinated with rate of carbon fixation. if too much sucrose/starch is made, the Calvin cycle stops. If too little sucrose/starch is made, Pi is tied up and Calvin cycle slows

Question 27

how is sucrose synthesis regulated with light?

Answer 27

fructose 2,6-bisphosphate production is increased in the dark by PFK-2, directing triose phosphates to glycolysis. light decreases the concentration of fructose 2,6-bisphosphate and gluconeogenesis is stimulated, leading to sucrose increase.
pt2 slide 16

Question 28

how is sucrose synthesis regulated by post translational modification?

Answer 28

Sucrose 6-phosphate synthase is less active when phosphorylated. SPS kinase (inhibited by glucose-6-phosphate) will phosphorylate SPS and SPS phosphatase (inhibited by Pi) will dephosphorylate. the active (dephosphorylated) SPS catalyzes the reaction that forms sucrose 6-phosphate.

Question 29

how is starch synthesis regulated with light?

Answer 29

in darkness ADP-glucose pyrophosphorylase is inhibited by Pi. in light ADP0glucose pyrophosphorylase is activated by 3-phosphpglycerate. This enzyme creates ADP-glucose, the precursor to starch.

Question 30

How is cellulose synthesized?

Answer 30

mechanism is not completely understood. it’s synthesized by structures called rosettes in the PM and requires a lipid primer. synthesis begins on one side of the membrane and flips to the other side.

Question 31

structures of sucrose, starch, cellulose, and peptidoglycan

Answer 31

sucrose: glucose (a1-2b) fructose
starch: glucose (a1-4) glucose
cellulose: glucose (b1-4) glucose
peptidoglycan: N-acetylglucosamine (b1-4) N-acetylmuramic acid linkages + peptide links to L-ala, D-ala, L-lys, and D-glut

Question 32

how is bacterial peptidoglycan synthesized?

Answer 32

its synthesized intracellularly but transported outside the PM. begins on one side of the membrane and the chain is flipped to other side
first step is activation of GlcNAc-1-phosphate with UTP. amino acids are added, then dolichol in PM, then 5 L-gly which serve as a cross link. Transpeptidase is the last enzyme that links two peptidoglycan chains together.
pt 2 slide 21

Question 33

how do penicillins work

Answer 33

penicillins are competitive inhibitors of cell wall synthesis. they have a B-lactam ring structure that looks like a peptide bond, tricking transpeptidase into binding. Once bound to penicillin, the transpeptidaase is inactivated. Some bacteria have B-lactamase which can inactivate penicillin and give antibiotic resistance

Question 34

what is vancomycin

Answer 34

used to be the last ditch effort antibiotic, but bacteria developed a resistance. it acts as a sponge and binds up the D-Ala-D-Ala dipeptide

Question 35

what are the aspects of carbohydrate metabolism that plants can do that animals cannot?

Answer 35

Fix CO2 (rubisco)
generate trioses (Calvin cycle)
convert acetyl-coA from FA to 4C compounds (glyoxylate)

Question 36

explain the glyoxylate cycle

Answer 36

occurs in seeds that don’t have access to light (no photosynthesis). plants convert acetyl-CoA from FA to glucose. acetyl-coA converts to succinate and leaves cycle. succinate can enter CAC to make oxaloacetate which can enter gluconeogensis
pt 2 slide 27

Question 37

what is the importance of have separate cellular compartments for glyoxylate cycle/B-oxidaton?

Answer 37

the glyoxysome keeps B-oxidation and glyoxylate enzymes separate from CAC enzymes (mitochondria) to prevent further oxidation of acetyl-CoA to CO2. instead it is used as precursor for gluconeogenesis.