cell bio exam 2: cellular metabolism

What are the two types of ATP synthesis pathways?

substrate-level phosphorylation: direct phosphate transfer (glycolysis, TCA cycle)

oxidative phosphorylation: indirect, powered by electron transfer chain and proton gradient

Where does glycolysis occur?

cytosol

What is the net ATP yield of glycolysis?

2 ATP

(4 ATP generate - 2 ATP consumed)

What is the net reaction of glycolysis?

C₆H₁₂O₆ + 2 NAD⁺ + 2 ADP^2- + 2 Pi^2- —> 2 C₃H₄O₄ (pyruvate) + 2NADH + 2 ATP

What is the purpose of glycolysis?

break glucose (6C) to 2 pyruvates (3C) while generating ATP & NADH (activated electron carriers)

What are the two phases of glycolysis pathway?

phase 1: energy investment - use 2 ATP to produce 2 G3P molecules

phase 2: energy payoff - produce 4 ATP, 2 NADH, and pyruvate

What are the 10 steps ot glycolysis?

phase 1 - energy investment

1. phosphorylation by hexokinase: [glucose + ATP → G6P + ADP]

2. rearrangement by PGI: [G6P ←→ F6P]

3. phosphorylation by PFK : [F6P + ATP → F1,6BP + ADP]

4. cleavage by aldolase: [F1,6BP (6C) ←→ DHAP (3C) + G3P (3C)]

5. isomerize by TPI: [DHAP ←→ G3P]

phase 2 - energy payoff

6. GAPDH oxidize + phosphorylate each G3P, reduce NAD⁺: [G3P + NAD⁺ + P ←→ 1,3-BPG + NADH +H⁺]

7. PGK phosphorylates ADP: [1,3-BPG + ADP ←→ 3PG + ATP]

8. rearrangement by PGM: [3-PG ←→ 2-PG]

9. removal of H₂O via. enolase: [2-PG ←→ PEP + H₂O]

10. PK phosphorylates ADP: [ PEP + ADP + H⁺ → pyruvate + ATP]

Which kinases are involved in glycolysis?

step 1: hexokinase (HK): ATP reactant, phosphorylate glucose

step 3: phosphofructokinase (PFK): ATP reactant, phosphorylate interrmediate F6P

step 7: phosphoglycerate kinase (PGK): phosphorylate ADP, ATP product

step 10: pyruvate kinase (PK): phosphorylate ADP reactant, ATP product

What does dehydrogenase do? Which dehydrogenases are involved in glycolysis?

dehydrogenase: remove H atom and transfers to electron carrier

step 6: GAPDH: remove H from G3P and adds H to NAD⁺ —> NADH

what does isomerase? which isomerases are involved in glycolysis?

isomerase: rearrange atom to form isomers

step 2: PGI - rearrange G6P to F6P

step 5: TPI - rearrange DHAP to G3P

what does mutase do? which mutases are involved in glycolysis?

mutase: type of isomerase that moves functional groups within molecules

step 8: PGM - rearrange 3-PG to 2-PG 

How do electron carriers work?

NAD⁺ accepts 2 electrons and 1 proton H⁺

reducing: NAD⁺ —> NADH; stores energy in unfavorable covalent bonds

oxidizing: NADH —> NAD⁺; break covalent bond to release energy

What are the two fates of pyruvate? How do ATP yield varies?

anaerobic: absence of O₂ → lactate or alcohol fermentation; 2 ATP

aerobic: presence of O₂ → transport to mitochondria matrix → pyruvate oxidation —> TCA cycle —> oxidative phosphorylation;  30-36 ATP

where does anaerobic respiration occur in the cell?

cytoplasm

what is the purpose of fermentation?

• does not generate any ATP

• regenerates NAD⁺ for glycolysis

How does lactic acid fermentation work?

LDH (lactase dehydrogenase) reduces pyruvate to lactate and oxidizes NADH to NAD⁺

[pyruvate (3C) + NADH —> lactate (3C) + NAD⁺]

How does alcohol fermentation work?

1. PDC (pyruvate decarboxylase) removes CO₂ from pyruvate to form acetaldehyde

[pyruvate (3C)—> acetaldehyde (2C) + CO₂]

2. ADH (alcohol dehydrogenase) reduces acetaldehyde to ehtanol and oxidizes NADH to NAD⁺

[acetaldehyde + NADH —> ethanol + NAD⁺]

What is glycolytic flux?

• rate of glucose to lactate turnover (ratio of 1 glucose : 2 lactate) 

• measures anaerobic respiration in RBC (since RBC has no mitochondria)

How is hexokinase allosterically inhibited?

excess G6P binds to allosteric site on hexokinase, causing a conformational change on active site that inhibits the reaction

What happens when there is no oxygen present during aerobic respiration?

no O₂ → TCA cycle and ETC turns off → [pyruvate] accumulates in mitochondrial matrix → no more pyruvate transported

How does oxidative decarboxylation of pyruvate work?

PDH (pyruvate dehydrogenase complex) oxidizes pyruvate, reduces NAD⁺ to NADH, and loses CO₂

[pyruvate (3C) + CoA + NAD⁺ → acetyl-CoA (2C) + CO₂ + NADH]

What happens during TCA cycle? How many TCA cycles per glucose?

• oxidize acetyl CoA and activate reduce 5 electron carriers (3 NADH, 1 FADH₂, 1 GTP) per cycle

• 2 CO₂ released

• 2 cycles per glucose

What are the steps of the TCA cycle?

1. citric synthase form citrate (6C); [acetyl-CoA (2C) + oxaloacetate (4C) → citrate (6C)]

2. aconitase rearranges: [citrate → isocitrate]

3. 1st oxidative decarboxylation by isocitrate dehydrogenase to release CO₂ and produce NADH: [isocitrate (6C) → a-ketoglutarate (5C) + CO₂ + NADH]

4. 2nd oxidative decarboxylation by a-ketoglutarate dehydrogenase to release CO₂ and produce NADH: [a-ketoglutarate (5C) + HS-CoA + NAD⁺ → succinyl-CoA (4C) + CO₂ + NADH]

5. substrate-level phosphorylation by succinyl-CoA synthetase: [succinyl-CoA + H₂O + P + GDP → succinate + GTP]

6. SDH (succinate dehydrogenase) oxidizes succinate and produces FADH₂: [succinate + FAD → fumarate + FADH₂]

7. fumarase adds H₂O to fumarate to form malate: [fumarate + H₂O → malate]

8. MDH (malonate dehydrogenase) regenerates oxaloacetate for next cycle: [malate + NAD⁺ → oxaloacetate + NADH]

What happens to all the glucose after aerobic respiration?

glucose gets fully oxidized to 6 CO₂ molecules during pyruvate oxidation and TCA cycle

where does oxidative phosphorylation occur?

inner mitochondrial membrane

What are the steps to oxidative phosphorylation?

1. electron donation & electron transport chain

2. proton pumping

3. O₂ final electron acceptor

4. proton motive force

5. ATP synthesis

How many electrons do the electron carriers donate during oxidative phosphorylation?

NADH: donates 2 electrons to Complex I

FADH₂: donates 2 electrons to Complex II

How do the electrons move in the electron transfer chain?

Complex I → Complex II → Ubiquinone → Complex III → Cytochrome C → Complex IV

How many protons do the complexes pump out? Where are the protons pumped to?

protons pumped from matrix → intermembrane space

Complex I and III: 4 protons

Complex IV: 2 protons

What is O₂ role in oxidative phosphorylation ?

• O₂ picks up 2 electrons and 2 protons to form H₂O

• occurs twice per 1 O₂ molecule

What are the two types of gradient?

• chemical gradient: pH difference

• electrical gradient: charge difference

Does reduced FADH₂ or NADH produce more ATP?

NADH, because pumps more protons.

NADH: complex II → Q → III →  IV

vs.

FADH₂: I → Q → III → C → IV

In cells that cannot carry out fermentation, which products derived from glycolysis will accumulate under anaerobic conditions?

pyruvate, NADH

Which steps of glycolysis are irreversible?

1. phosphate group transfer from ATP to glucose → G6P

3. phosphate group transfer from ATP to F6P → F1,6-BP

10. phosphate group transfer from PEP to ADP → pyruvate + ATP

Which carbons are released from TCA cycle?

two carbons from oxaloacetate

Explain Kreb’s experiment that supports that the citric acid cycle is cyclical instead of linear

malonate = inhibitor

when fumarate is added

if linear: increase [oxaloacetate]

bc cyclical: no change [oxaloacetate], increase [succinate]

What happens to the acetyl CoA carbons after one round of TCA cycle?

regenerates to oxaloacetate

How does phosphofructokinase (PFK) regulate glycolysis?

• rate-limiting step

• commitment step to glycolysis

• inhibitors: ATP (allosteric inhibitor), citrate (signals abundant carbon)

• activators: AMP, ADP (high conc. when ATP conc. is low)

Which molecules are required for the citric acid cycle to fully oxidize the carbons donated by acetyl CoA?

• NAD+

• GDP

• O2

• oxaloacetate

What are the two structures of ATP synthase?

F_o (membrane part): forms channel for protons (H⁺) to flow through

F₁ (matrix/cytoplasmic part): where ATP is synthesized

What is proton-motive force?

stored energy from electrochemical gradient

How does ATP synthase work?

1. protons flow back into matrix via. F₀ channel

2. F₀ rotate mechanically, inducing series of conformational changes to F₁ to bind ADP + Pi → ATP and release ATP

What happens when there is excess ATP?

ATP synthase turns off → increase [H⁺] in IMS → ETC backed up → NADH + FADH₂ accumulation → NADH + FADH₂ allosterically inhibit TCA cycle + glycolysis → aerobic pathway turns off

What molecules stimulates vs. inhibits cellular respiration?

stimulates (oxidized electron carrier) - ADP, NAD+, FAD

inhibits (reduced electron carriers) - ATP, NADH, FADH2

How do pyruvate and inorganic phosphate move through the cell membrane?

move into the matrix w/ protons as they move down their electrochemical gradient

Where does ADP vs. ATP go?

ADP - pumped into matrix

ATP - pumped out of matrix

antiport process

What does insulin do?

• removes glucose from blood and transfers into cells

• stimulates glycolysis (step 1 phosphorylating to G6P)

What is glycogen synthetase? How is it actived?

function: make glycogen

allosteric activation: excess G6P → glycolysis slows down → glycogen synthetase activates and store excess G6P as glycogen

What is glycogen phosphorylase? How is it allosterically inhibited?

• breaks apart glycogen

• excess G6P inhibits glycogen phosphorylase

What are the two pathways when there is no glucose?

glycogenolysis (<1 day fasting)

beta-oxidation (stores ~1 month)

How does glycogenolysis work?

1. glycogen broken down to G6P via. glycogen phosphorylase

2. G6P is broken down to free glucose by glucose-6-phosphatase and released in blood

3. free glucose enter cells and undergoes glycolysis

How does fatty acid oxidation work?

1. break down triglycerides into free aftty acids and glycerol

2. beta oxidation: oxidize fatty acids to acetyl-CoA, reduce NAD⁺ to NADH, reduce FAD to FADH₂

How does gluconeogenesis occur?

glycolysis in reverse, except irreversible steps need diff enzymes (1, 3, 10)

Where does the input of ATP and pyruvate come from for gluconeogenesis?

ATP - fatty acid metabolism

pyruvate - from amino acids

How does excess ATP concentration turn off ETC directly?

increase [ATP] = decrease [ATP]

so ATP synthase shuts off/reverse spins

increase in [H+] in IMS

ETC turns off bc no longer need to pump protons

How does excess ATP concentration turn off glycolysis directly?

ATP is direct inhibitor of enzymes in glycolysis (PFK, PK)

What happens when there is a loss of function of antiport?

How does this affect aerobic respiration?

How does it affect anaerobic respiration?

• ATP stays in matrix, cant be transported out to the cytoplasm

• ADP stays in cytoplasm, can’t be transported into matrix

inhibit aerobic — high ATP conc. will shut off ATP synthase

stimulate anaerobic — ATP generated from glycolysis would be in cytoplasm

What happens to the consumed carbon atoms during oxidation of glucose?

What about oxygen?

carbons → released as CO₂

oxygen → released as H₂O