Chapter 16 (Cont.) -20 Flashcards
1
Q
Step 1: Condensation
- What is the reaction?
- What is the enzyme?
- What goes into the reaction? What comes out?
- What is the activity of this reaction largely dependent on?
- Is it thermodynamically favorable/irreversible?
- What is it regulated by?
- How is citrate synthase unique?
A
- Acetyl-CoA + Oxaloacetate ⇒ Citrate
- Citrate Synthase, -32.2 kJ/mol, Rate determining step.
- H2O goes in, CoA-SH comes out.
- Largely dependent on Oxaloacetate
- Highly thermodynamically favorable.
- Regulated by substrate availability and product inhibition.
- It has an induced fit.
2
Q
Step 2:
- What is the reaction?
- What is the enzyme?
- What goes in? What comes out?
- What does the elimination of water yield?
- Why must citrate be converted into isocitrate?
- Is the reaction thermodynamically favorable/irreversible?
- Water must be removed from citrate and then subsequently added to cis-aconitate. What is this catalyzed by?
- Only one version of isocitrate is produced by aconitase. What is this version?
A
- Citrate ⇔ Isocitrate
- Aconitase, 13.3 kJ/mol
- Water is released, then added to the formed intermediate.
- Gives a C-C double bond.
- Citrate, a tertiary alcohol, is a poor substrate for oxidation. Isocitrate, a secondary alcohol, is a good substrate for oxidation.
- Thermodynamically unfavorable/reversible. Product concentration kept low to pull forward.
- Only R-isocitrate is produced by aconitase. Distinguished by three-point attachment to the active site.
3
Q
Step 3
- What is the reaction?
- What is the enzyme?
- What is made at this step? What is released?
- What is the alcohol oxidized to?
- What kind of enzymes use NADP+ as a cofactor?
- Is the reaction thermodynamically favorable/irreversible?
A
- Isocitrate ⇒ α-Ketoglutarate + CO2
- Isocitrate dehydrogenase, -20.9 kJ/mol
- NADPH is made, CO2 is released
- Alcohol is oxidized to a ketone. Transfers a hydride to NAD.
- Cytosolic isozymes.
- Highly thermodynamically favorable. Regulated by ATP and product inhibition.
4
Q
Step 4
- What is the reaction?
- What is the enzyme?
- What is made at this step? What is released?
- After two turns of the cycle where do the carbons come from?
- What is unique about the product?
- Is the reaction thermodynamically favorable/irreversible?
- What is α-Ketoglutarate dehydrogenase?
A
- α-Ketoglutarate ⇒ Succinyl-CoA
- α-Ketoglutarate dehydrogenase
- NADH produced, CO2 released
- Carbons lost now come from oxaloacetate.
- Succinyl-CoA is a high energy thioester bond.
- Highly thermodynamically favorable/irreversible. Regulated by product inhibition.
- Complex similar to pyruvate dehydrogenase. Same coenzymes, identical mechanisms. Active sites different to accommodate different-sized substrates.
5
Q
Step 5: Formation of GTP
- What is the reaction?
- What is the enzyme?
- What is made at this step? What is released?
- What kind of phosphorylation occurs at this step?
- What does the energy of the thiolester allow for?
- What is produced? What can it be converted to?
- Is the reaction thermodynamically favorable/irreversible?
A
- Succinyl-CoA ⇔ Succinate
- Succinyl-CoA synthetase, -2.9 kJ/mol
- GTP is made from GDP + Pi, CoA-SH is released
- Substrate level
- Allows for incorporation of inorganic phosphate.
- Produces GTP, which can be made into ATP.
- Slightly thermodynamically favorable/reversible. Product concentration kept low to pull reaction forward.
6
Q
Step 6
- What is the reaction?
- What is the enzyme?
- What is made at this step? What is released?
- What is covalently bound to succinate dehydrogenase?
- What does FADH2 pass its electrons to?
- Where is this enzyme located? What is it part of?
- Is the reaction thermodynamically favorable/irreversible?
A
- Succinate ⇔ Fumarate
- Succinate dehydrogenase
- FADH2 is made at this step, 0 kJ/mol
- FAD is covalently bound
- To coenzyme Q
- In the inner mitochondrial membrane, part of Complex II of the electron transport chain.
- Near equilibrium/reversible. Product concentration kept low to pull forward.
7
Q
Step 7
- What is the reaction?
- What is the enzyme?
- What is made at this step? What is released?
- How is this reaction sterospecific?
- Is the reaction thermodynamically favorable/irreversible?
A
- Fumarate ⇔ Carbanion transition state ⇔ L-Malate
- Fumarase
- -3.8 kJ/mol
- Addition of water is always trans and forms L-malate. OH- adds to fumarate… then H+ adds to the carbanion.
- Slightly thermodynamically favorable/reversible. Product concentration kept low to pull reaction forward.
8
Q
Step 8
- What is the reaction?
- What is the enzyme?
- What is produced?
- What is regenerated at this step?
- Is the reaction thermodynamically favorable/irreversible?
A
- L-Malate ⇔ Oxaloacetate
- Malate dehydrogenase, 29.7 kJ/mol.
- NADH is produce.
- Oxaloacetate is regenerated for citrate synthase.
- Highly thermodynamically unfavorable/reversible. Oxaloacetate concentration kept VERY low by citrate synthase. Pulls the reaction forward.

9
Q
- What are anaplerotic reactions?
- Where is the citric acid cycle regulated?
- What are the general regulartory mechanisms?
A
- Intermediates in the citric acid cycle can be used in biosynthetic pathways (removed from cycle).
- Must replenish the intermediates in order for the cycle and central metabolic pathway to continue.
- 4-carbon intermediates are formed by carboxylation of 3-carbon precursors.
- Regulated at highly thermodynamically favorable and irreversible steps.
- PDH, citrate synthase, IDH, and KDH
- Regulatory Mechanisms
- Activated by substrate availability
- Inhibited by product accumulation
- Overall products of the pathway are NADH and ATP.
- Affect all regulated enzymes in the cycle
- Inhibitors: NADH and ATP
- Activators: NAD+ and AMP
10
Q
- How is pyruvate dehydrogenase regulated at E1?
- How do PDH kinase and PDH phosphorylase work?
A
- Regulation of E1:
- Phosphorylation: inactive
- Dephosphorylation: active
- Are part of mammalian PDH complex.
- Kinase is activated by ATP.
- High ATP → phosphorylated PDH → less acetyl-CoA.
- Low ATP → kinase is less active and phosphorylase removes phosphate from PDH → more acetyl-CoA
- Kinase is activated by ATP.
11
Q
- What is citrate synthesis also inhibited by?
- What is an important branch point for amino acid metabolism?
- What role does Succinyl-CoA play?
- What controls citrate levels?
- What does inhibition of IDH lead to?
A
- Citrate synthase is also inhibited by succinyl-CoA.
- α-ketoglutarate is an important branch point for amino acid metabolism.
- Succinyl-CoA communicates flow at this branch point to the start of the cycle.
- Regulation of isocitrate dehydrogenase controls citrate levels.
- Inhibition of IDH leads to accumulation of isocitrate and reverses acconitase. Accumulated citrate leaves mitochondria and inhibits phosphofructokinase in glycolysis.
12
Q
- How are plants versatile in biosynthesis?
- What kind of pathway is needed for carbohydrate biosynthesis in plants?
- Where do these reactions occur?
A
- Plants are extremely versatile in biosynthesis.
- Can build organic compounds from CO2.
- Can use energy of sunlight to support biosynthesis.
- Can adopt to a variety of environmental situations.
- Anabolic pathway: Uses ATP (energy) and NADPH (electrons) to convert CO2 into sugars.
- In plants: ATP is generated from light/photon absorption.
- NAPDH is generated by oxidation of H20 to O2 using light.
- Reactions occur in a specialized organelle, the chloroplast.
13
Q
- What kind of experiment did Calvin carry out?
- What was his first observation. What was the interpretation of this observation?
- What was his second observation. What was the interpretation of this observation?
A
- Incubated green algae with 14CO2 and traced the metabolic fate of 14C.
- First observation: within less than a minute, 14C-labeled amino acids and sugars were found → green algae are able to convert CO2 into small organic compounds (“CO2-assimilation”).
- Within 5 sec of incubation in 14CO2, labeled 3-phosphoglycerate (3-PG) was detected (C3-plants).
- 3PG is a stable intermediate.
- 3PG is formed by carboxylation of carbon intermediate.
14
Q
- What do autotrophic organisms use CO2 as a source of?
- How is CO2 reduced to carbon intermediates?
A
- Use CO2 as sole source for biosynthesis of starch, cellulose, lipids and proteins and other organic molecules.
- Use reducing equivalents of NADPH and energy (ATP), which is generated during photosynthesis to reduce CO2 to carbon intermediates.
15
Q
- What does three turns of the Calvin Cycle generate?
Step 1: CO2-Fixation
- What does this reaction consist of?
- What is the enzyme used?
- What does the active site of this enzyme contain?
- What is it coordinated by?
A
- 3 CO2, 9 ATP, 6 NADPH. All converted into 1 glyceraldehyde-3-phosphate.
Step 1: CO2-Fixation
- Carboxylation of ribulose 1,5 B-P to generate 2 molecules of 3-PG (catalyzed by ribulose 1,5 B-P carboxylase.
- Catalyzed by Rubisco - most abundant protein in the biosphere.
- Active site contains Mg2+
- Mg2+ is coordinated by 3 AA side chain oxygens, including a carbamoylated Lys (-NH-COO-). Mg2+ forms coordinative bonds with both substrates, CO2 and Ru 1,5bP. Mg2+ orients and polarizes the substrates.
16
Q
Step 2: Reduction
- What is the reaction?
- How does this step related to glycolysis?
- What cofactor is used?
- What can glyceraldehyde-3-P be used for?
- How is NADPH different from GAPDH?
- What is used to regenerate Ru 1,5 bP?
A
- 3-phosphoglycerate reduced to glyceraldehyde-3-P
- Reversal of glycolysis
- NADPH is being used instead of NADH, ATP is also used during this step.
- Uses of glyceraldehyde-3-P
- Stored as starch in chloroplast for later use.
- Translocated to cytosol and converted to sucrose (transported to non-photosynthesizing parts).
- Unlike GAPDH from cytoplasmic gluconeogenesis, stromal enzyme uses NADPH as co-factor.
- Majority of GA 3P is used to regenerate Ru 1,5 bP.

17
Q
Step 3: Regeneration
- What is the reaction?
- What step is this similar to in the PPP?
- What is the goal of this step?
- What are the pentose phosphates converted into?
- What phosphorylates Ru 5P → Ru 1,5 bP?
- What is the net reaction of the Calvin Cycle?
- What is the stochiometry problem?
A
- Regeneration of ribulose 1,5 bisphosphate starting from fruc-6-P.
- Very similar to the non-oxidative part of the pentose phosphate pathway except that it proceeds in the opposite direction = reductive pentose phosphate pathway (from hexose to pentose).
- Conversion of five three-carbon compounds (15 carbons) into three five-carbon compounds (15 carbons).
- Converted into Ru 5P
- Ru 5P kinase
- Picture
- The cycle will eventually run out of inorganic phosphate.

18
Q
- What does the Pi/Triose Antiport do?
- What else is this antiport used for?
A
- Export of triose phosphates for sucrose synthesis in the cytosol would soon deplete Pi pool in the chloroplast stroma → Pi -neutral antiport.
- Antiport is also used to transfer NAD(P)H and ATP produced by photosystems into the cytosol.

19
Q
- What does exposure to light produce?
- What happens when [H+] is pumped from the stroma into the thylakoid?
- What does high pH and Mg2+ in the stroma mean?
A
- Exposure to light → light reaction produces ATP and NADPH required for CO2 assimilation.
- [H+] is pumped from stroma into thylakoid → Mg2+ is exported in exchange.
- High pH and [Mg2+] in stroma
- Promote carbamoylation of Rubisco → activation.
- Activate chloroplast Frc 1,6bPase
20
Q
Calvin Cycle: Redox
- What does light exposure lead to?
- What does Fdred reduce?
- What happens in the dark?
A
- e- transfer from water → ferredoxin → NADP+
- Reduces thioredoxin (Trx), which in turn reduces S-S bonds in several enzymes of Calvin cycle → activation.
- O2 re-oxidizes SH groups and inactivates enzymes.
21
Q
- Why is there deleterious side-reactions of Rubisco?
- How often do these reactions occur?
- What how is this process challenging for plants in hot climates?
- How is the production of 2-Phosphoglycolate salvaged?
A
- Ru 1,5bP reacts with O2 instead of CO2.Consumes CO2 acceptor Ru 1,5 bP without achieving CO2 assimilation.
- One in every three or four turnovers – it forms 3-Phosphoglycerate and 2-Phosphoglycolate.
- Photorespiration increases with temperature.
- 2-Phosphoglycolate can undergo further reactions to to yield one triosephosphate and a CO2 but no energy is produced. Instead it consumes a lot of energy.
22
Q
- How do C3 plants differ from C4 plants?
- What is an additional problem in hot dry climates?
- What do CAM plants do?
- What happens at night?
A
- Plants discussed so far are called C3 plants: CO2-fixation results in formation of C3 compound (3-Phosphoglycerate). C4 plants: CO2-fixation and Rubisco activity are spatially separated (2 different cell types). Mesophyll cells: CO2 (HCO3-) is first used to synthesize oxaloacetate (C4 compound).
- Gas exchange via stomata (CO2 in, O2 out) results in loss of precious water.
- CO2-fixation (C4 cycle) and Rubisco activity occur in the same cell, but are temporally separated (2 different times).
- Stomata are open, CO2 (HCO3-) is used to form oxaloacetate → malate.