Glycolysis, pyruvate oxidation, Krebs cycle Flashcards by LV D

What is the function of glycolysis?

Phases?

Where does it happen?

1 molecule of glucose is converted to 2 molecules of pyruvate, in 3 phases - 10 steps:

energy investment: 3 steps
cleavage of C6 sugar to 2 C3 sugars: 2 steps
energy generation: 5 steps

happens in cytosol

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What are the 2 net equations of glyclosis?

Why plural?

can happen either under aerobic or anaerobic conditions

aerobic: cf. image
anaerobic: 2 NAD⁺ → 2 NADH/H⁺ less
(→ NADH⁺ used to produce lactate)

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What would reasons for anaerobic conditions be?

Examples.

hypoxia/anoxia due to resp./circ. pathologic conditions, asphyxia, poisons
relative hypoxia due to extreme O₂ consumption, e.g. during exercise
lack of mitochondria, e.g. in RBCs (→ always anaerobic)

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What is the 1^st step of glycolysis?

Catalyzed by.. ?

Anything important?

Structures.

hexokinase/glucokinase
1^st ATP used

glucose + ATP → glucose-6P + ADP

IRREVERSIBLE, inhibited allosterically by product

_{part of energy generation phase}

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What is the difference btw hexo- and glucokinase?

Effects?

hexokinase: low K_m for glucose, always active
glucokinase: high K_m, active after meal to remove glucose from hepatic portal blood
- in liver → energy
- in pancreatic β-cells → detect [glucose], triggers insulin release

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What happens to G6P?

Catalyzed by.. ?

Structures.

phosphohexose isomerase
isomerizes aldose to ketose

glucose-6P ⇔ fructuose-6P

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What happens to F6P?

Catalyzed by.. ?

Anything important?

Structures.

phosphofructokinase 1
2^nd ATP used

F6P + ATP → fructose 1-6-bisphosphate

IRREVERSIBLE, rate-limiting step of glycolysis

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What happens to F1,6BP?

Catalyzed by.. ?

Structures.

aldose A
cleaves F1,6BP into 2 trioses

F-1,6-BP → G3P + DHAP

NOTE: only G3P can proceed immediately through glycolysis

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What happens to DHAP?

Catalyzed by.. ?

Structures.

phosphotriose isomerase

DHAP ⇔ G3P

necessary b/c DHAP cannot proceed further in glycolysis w/o isomerization → after this step: 2 G3P

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What happens to G3P?

Catalyzed by.. ?

Structures.

glyceraldehyde 3-P dehydrogenase
anhydride bond formed

G3P + NAD⁺ + P_i → 1,3BPG + NADH

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Which substance is an inhibitor of glyceraldehyde 3P dehydrogenase?

iodoacetate can bind to -SH groups, causing inhibition

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What happens to 1,3BPG?

Catalyzed by.. ?

Structures.

phosphoglycerate kinase

1,3-BPG + ADP ⇔ 3-phosphoglycerate + ATP

1^st substrate level phosphorylation

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1,3BPG can normally be used for glycolysis in erythrocytes or… ?

Catalyzed by?

Name the enzyme catalyzing the reverse reaction, too.

bisphosphoglycerate mutase

1,3BPG ⇔ 2,3BPG
(instead of synthesis of 3-phosphoglycerate)

BUT: can be converted to 3-phosphogylcerate by 2,3-BPG phosphatase (w/o ATP yield)

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Why is arsenic toxic and lethal?

analogue of P_i→ energy in ester/anhydride bonds lost

⇒ uncoupling oxidation and phosphorylation in glycolysis

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What happens to 3-phosphoglycerate?

Catalyzed by.. ?

Structures.

phosphoglycerate mutase
moves position of ester bond

3-phosphoglycerate ⇔ 2-phosphoglycerate

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What happens to 2-phosphoglycerate?

Catalyzed by.. ?

Structures.

enolase
dehydration to form enol (macroergic bond)

2-PG ⇔ PEP + H₂O

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What compounds act as cofactor for enolase?

Mg²⁺, Mn²⁺

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Which substance can inhibit enolase?

fluoride

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What happens to PEP?

Catalyzed by.. ?

Anything important?

Structures.

pyruvate kinase

PEP + APD → pyruvate + ATP

2^nd substrate level phosphorylation

+ irreversible b/c pyruvate isomerizes from high E enol into keto-form

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What are possible pathways of the 2 molecules of pyruvate produced in glycolysis?

under aerobic conditions: enter PDC, are converted to 2 acetyl-CoA, 2 CO₂ released
under anaerobic conditions: LDH converts both pyruvate to 2 lactate, 2 NADH consumed

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What is the function of the pyruvate dehydrogenase complex (PDC)?

Where does it happen?

oxidative decarboxylation: pyruvate → acetyl-CoA

happens in mitochondrial matrix
irreversible

(requires O₂ = only aerobic, releases CO₂)

What is the net equation of the reaction catalyzed by PDC?

pyruvate + NAD⁺ + CoA-SH

↓

acetyl-CoA + NADH/H⁺ + CO₂

Describe the structure of the PDC, listing subunits, and their prosthetic groups.

3 subunits:

E₁: pyruvate dehydrogenase (TPP = prost. group)
E₂: dihydrolipoyl transacetylase (lipoic acid = prost. group)
E₃: dihydrolipoyl dehydrogenase (FAD = prost. group)

_{TPP (thiamine <span>p</span>yrophosphate) and TDP (<span>d</span>iphosphate) are same thing}

Which substances act as coenzymes during oxidative carboxylation of pyruvate?

NAD⁺ and CoA-SH

Which reaction is catalyzed by E₁ of the PDC? Name of enzyme. Structures.

**_pyruvate dehydrogenase_** oxidative phosphorylation **pyruvate + TPP → hydroxyethyl-TPP + CO₂** _{TPP (thiamine pyrophosphate) and TDP (diphosphate) are same thing}

What is another name for thiamin? What are possible consequences of a thiamin deficiency? Why?

**vitamin B₁** * deficiency can be caused by _alcoholism_ (poor diet + reduced thiamin absorption) * leads to _impaired glucose metabolism_, possible life-threatening _lactic/pyruvic acidosis_ (bc part of TPP)

What is the first reaction catalyzed by E₂ of the PDC? Name of enzyme Structures.

**_dihydrolipoyl transacetylase_** oxidized lipoamide + hydroxyethyl-TPP → acetyl-lipoamide + TPP _{TPP (thiamine pyrophosphate) and TDP (diphosphate) are same thing}

What is the second reaction catalyzed by E₂ of the PDC? Name of enzyme. Structures.

**_dihydrolipoyl transacetylase_** acetyl dihydrolipoamide + CoA-SH → dihydrolipoamide + acetyl-CoA _{TPP (thiamine pyrophosphate) and TDP (diphosphate) are same thing}

Which reaction is catalyzed by E₃ of the PDC? Name of enzyme Structures.

**_dihydrolipoyl dehydrogenase_** dihydrolipoamide + FAD → oxidized lipoamide + FADH₂ → then transferred to NAD⁺ → _NADH_ formed

How do you call the high energy bond in acetyl-CoA?

**thioester**

As a summary... List the products of each step of the oxidation of pyruvate.

pyruvate converted to 1. E₁ → **hydroxyethyl-TPP + CO₂** 2. E₂ (step 1) → **acetyl-dihydrolipoamide** + **TPP** 3. E₂ (step 2) → **dihydrolipoamide + acetyl-CoA** 4. E₃ → **oxidized lipoamide + NADH/H⁺ + FAD** ****⇒ overall: acetyl-CoA + CO₂ + NADH/H⁺

What are the 2 mechanisms of regulation of the PD complex?

* **allosteric inhibition:** E₂ and E₃ subunits * **de-/phosphorylation:** of E₁ subunit by _PDC kinase_ and _phosphatase_

Which subunits of PDC are allosterically inhibited? By which substances?

product inhibition * _E₂ inhibited by:_ **acetyl-CoA** * _E₃ inhibited by:_ **NADH**

Which subunit of PDC is regulated by de-/phosphorylation? By which substances?

E₁ either _phosphorylated = inhibited by **PDC kinase**_ * lot of energy: **ATP**, **NADH** * product: **acetyl-CoA** _dephosphorylated = activated by **PDC phosphatase**_ * **insulin** * exercise: **Ca²⁺, Mg²⁺**

What does dichloroacetate do?

**inhibits PDC kinase** → no phosphorylation, PDC active

Which molecules serve as substrate of the Krebs cycle, what are the final products? Where does it happen? Aerobic or anaerobic?

_oxidation:_ acetyl-CoA → 2 CO₂ ⇒ 1 ATP equiv., 1 FADH₂, 3 NADH produced, used for ox. phosphorylation * happens in **mitochondrial matrix** * only in **aerobic conditions** 8 steps

What is anaplerosis?

*ex:* intermediates in Krebs cycle intermediates are all **resynthesized, not consumed**

What happens to acetyl-CoA (first step of the Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_citrate synthase_** via intermediate **acetyl-CoA + OXA + H₂O → citrate + CoA-SH** **irreversible** b/c thioester bond of acetyl-CoA cleaved

What happens to citrate (second step of the Krebs cycle)? Catalyzed by.. ? This is the reason why... ? Structures.

**_aconitase_** citrate dehydrated to intermediate, eventually isomerized to isocitrate **citrate ⇔ H₂O + cis-aconitate ⇔ isocitrate** ⇒ reason why first CO₂s lost, not carbon atoms of acetyl-CoA (but OXA..?)

Which substance can inhibit the aconitase?

**thoroacetate**

What happens to iscitrate (third step of the Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_isocitrate dehydrogenase_** **isocitrate + NAD⁺ → CO₂ + NADH + α-KG** * 1^st oxidative decarboxylation * irreversible

What is the cofactor of isocitrate DEH?

Mg²⁺/Mn²⁺

What happens to α-KG (fourth step of the Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_α-ketoglutarate dehydrogenase complex_** **α-KG + CoA-SH + NAD⁺ → CO₂ + NADH + succinyl-CoA** * 2^nd oxidative decarboxylation * irreversible similar to PDC, same E₃ subunit, uses same cofactors

Which substance/condition inhibit the α-ketoglutarate dehydrogenase complex?

* **arsenite** * **hyperammonemia**

What happens to succinyl-CoA (fifth step of Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_succinyl-CoA synthetase_** **succinyl-CoA + H₂O + GDP + P_i ⇔ succinate + GTP + CoA-SH** * substrate-level phosphorylation * in gluconeogenic tissues (liver/kidney) ATP _can_ be formed instead of GTP

What happens to succinate (sixth step of Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_succinate dehydrogenase_** **succinate + FAD ⇔ FADH₂ + fumarate** on _inner mit. membrane_ (complex II), e^- from FADH transferred via Fe-S centers to CoQ

Which substance inhibits succinate dehydrogenase?

**malonate** binds _competitively_ to succinate dehydrogenase

What happens to fumarate (7th step of Krebs cycle)? Catalyzed by.. ? Structures.

**_fumarase_** **fumarate + H₂O ⇔ L-malate**

What happens to malate (8th step of Krebs cycle)? Catalyzed by.. ? Anything important? Structures.

**_malate dehydrogenase_** **malate + NAD⁺ → OXA + NADH** **BUT:** high [reagents] strongly shift equlibrium to the right (favoring products, irreversible) _{NOTE: equilibrium inversed in case of gluconeogenesis}

How is the activity of the Krebs cycle regulated?

happens only in **aerobic conditions**, activated by * **low [NADH],** **[ATP]** * **low [succinyl-CoA]** * **Ca²⁺** = e.g. during exercise

Which vitamins act as coactivators, being essential in the Krebs cycle?

_4 B vitamins_ * **riboflavin:** in form of FAD * **niacin:** in form of NAD⁺ * **thiamin:** as TPP, part of α-ketoglutarate dehydrogenase complex * **pantothenic acid:** part of CoA

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Glycolysis, pyruvate oxidation, Krebs cycle Flashcards

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