TCA cycle, AA biosynthesis, intermediate catabolism and anabolism (2-3)

why do we need to add CoA to oxidize? Why can’t we just use oxidize acetate?

because we wouldn’t be able to directly oxidize CH4

how many electrons do we gain from one round of the TCA cycle?

8

• 4 pairs of electrons→ 1 on NADPH, 1 on FADH2, 2 on NADH

how many pairs of electrons are harvested from each pyruvate?

5 pairs (10 e-)

• add an NADH from pyruvate dehydroxogenase rxn

how many pairs of electrons per glucose?

10 pairs

how many electron pairs of EMP glycolysis plus TCA cycle?

12 pairs

• 2 pairs from glycolysis

• 10 pairs from TCA cycle

how many electron pairs from PPP → TCA?

12 pairs

• 6 pairs for (3x glucose getting oxidized releasing 2 NADH)

• 1 from payoff phase of glycolysis

• 5 from TCA cycle

how many electron pairs from ED → TCA?

12 pairs

• 1 pair from 1st part of Ed→ NADPH

• 1 from payoff phase of glycolysis

• 10 from TCA cycle for the two pyruvate

1st step of TCA

oxaloacetate+Acetyl-CoA+H2O→ Citrate+CoA-SH

• citrate synthase

• powered by hydrolysis of acetyl-CoA thioester bond

• very exergonic

citrate synthase

• transferase

• oxaloacetate+Acetyl-CoA+H2O→ Citrate+CoA-SH

• oxaloacetate needs to bind to citrate synthase first to open a binding pocket for acetyl-CoA

step 2 and 3 of TCA cycle

overall: citrate→ isocitrate

2. citrate→ H2O+cis-aconitate

3. cis-aconitate+H2O→ isocitrate

• moves -OH from C4 to C5

why do we do steps 2 and 3?

to move the OH bond to destabilize the bond so we can make an easy cleavage

aconitatse

Citrate→ isocitrate

• isomerase

delta G of steps 2 and 3

pretty positive, but since step 1 is so negative, we have a TON of citrate, so we need to get rid of having so much more to just more compared to isocitrate

iron sulfur center

cofactors in the enzyme that help pull of and add H2O in steps 2 and 3

steps 4 and 5 of TCA cycle

isocitrate→ [oxalosuccinate]→ \alpha-ketoglutarate + CO2

• irreversible

isocitrate dehydrogenase (IDH)

oxidoreductase

1. oxidizes isocitrate → [oxalosuccinate] +NADPH + H

2. → C5=O attracts electrons from C4→ destabilizing C4-C3 bond → release C3 as CO2

3. [oxalosuccinate]→ CO2+a-ketoglutarate

main isozyme for TCA cycle?

dehydrogenase 3 (generate NADH)

step 6 of TCA cycle

a-ketogluterate + CoA-SH+ NAD+→ succinyl-CoA+CO2+NADH

• super exergonic

• irreversible

a-ketoglutarate dehydrogenase complex

a-ketogluterate + CoASH→ NADH + CO2 + succinyl-CoA

1. E1: a-ketogluterate binds → decarboxylated then thrown on a lipoic acid

2. E2: remaining 4 Cs get transferred to CoA then leave

3. E3: lipoic acid gets reduced → FADH2→ gets oxidized and puts electrons on NAD+→ NADH

• same thing as pyruvate dehydrogenase

step 7 of TCA

succinyl-CoA→ Succinate

• gives off: GTP (or ATP) and CoA-SH

succinyl-CoA synthetase (succinic thiokinase)

succinyl-CoA→ Succinate

• ligase

• cleaves bond of S-CoA on C3→ rearrange C3-(C4)OO-

• substrate level phosphorylation (Pi+GDP→ GTP)

succinyl-CoA synthetase reaction (3 steps)

1. switch out S-CoA with O-Pi

2. histidine takes Pi off succinate → O-

3. histidine puts Pi on whatever is needed (GDP or ADP)

nucleoside diphosphate kinase

catalyzes the reversible conversion of GTP and ATP

• GTP+ADP\lrArr GDP+ATP

step 8 of TCA

succinate→ fumerate

succinate dehydrogenase

• membrane bound (has to happen in membrane because products are part of electron transport chain)

• puts electrons of FAD→ FADH2→ electrons go to UQ (because FADH2 can’t leave, it’s a prosthetic group)

• creates methylene group (double bond between C2=C3)

step 9 of TCA

fumerate→ malate

• reversible

fumarase

step 9: fumarate→ L-malate

• lyase

• adds water → goes for C2=C3 → OH on C2

step 10 of TCA

L-Malate→ Oxaloacetate

• transfers 4th pair of e-

• super endergonic

L-malate dehydrogenase

malate→ oxaloacetate

• oxidoreductase

• transfers e- to NAD+→ NADH

• goes from (C2)-OH→ C2=O

• super endergonic, but goes in forward reaction because there is like NO oxaloacetate

metabolons

multienxyme complexes that are close together and the exit site of one enzyme is right next to the entrance site of another

held together by non covalent interactions

amphibolic

something that is both catabolic and anabolic

other inputs and outputs of respiration (4)

1. fatty acids \lrArr acetyl-CoA

2. penrose sugars \lrArr PPP

3. N of nitrogenous bases \lrArr AAs metabolism

4. AAs feed in and are made from PPP, PEP, pyruvate, and TCA intermediates

ways to make Glu (2)

1. at high NH3→ NH3 directly added while a-ketoglutarate is reduced

2. at low NH3→ a-ketoglutarate transaminated by Gen to 2 Glu

Glen synthase

regenerates Glen from Glu

glutamate dehydrogenase

high Km for NH3 (only works at high NH3)

• reduces a-ketoglutarate→ L-glutamate

• adds NH3

what is the Cell’s entry point for NH3?

Gln synthase (unless NH3 is very high)

transaminations using Glu as donor

• pyruvate → alanine

• oxaloacetate→ aspartate

• backbone

transaminations using Glu as donor

• side chains transfer: aspartate→ asparagine

put NH2 on aketoglutarate…

makes glutamate

EMP intermediates used for AA synthesis

pyruvate, PEP, 3-PGA

-9 AAs

TCA intermediates used for AAs

oxaloacetate and a-ketoglutarate

• 10 AAs

how do fermenters generate intermediates from the TCA cycle?

1. have an oxidative branch that make a-ketogulatarate

2. have a reductive branch that helps with reductive pressures (makes succinyl-CoA)

   1. 2 pairs of e- consumed (makes up for produced in oxidative branch)

   2. use fumerate reductase to make succinate

anaplerotic reactions

reactions that replenish the intermediates

pyruvate carboxylase

put CO2 on pyruvate → oxaloacetate

• same one used in gluconeogensis

biotin

coenzyme used as a carrier of 1 C groups like CO2 (used in pyruvate→ OAA)