PDH & CAC Flashcards
Pyruvate dehydrogenase complex
Composed of 3 enzyme subunits in mitochondria thatcatalyzes the oxidative decarboxylation of pyruvate into Acetyl-CoA, CO2, NADH
Oxidation of FA, glucose, aa, acetate, and ketone bodies all generate ___, which is the substrate for the CAC
Acetyl-CoA
Acetyl CoA produced in the PDH complex or from the oxidation of FA or aa enters the ____ . What happens?
CAC
Oxidize the acetyl-CoA
Form more NADH & FADH2
What happens in the ETC?
- NADH and FADH2 get oxidized and the electrons are donated to the ETC, where the final electron acceptor is O2 –> H2O
- Meanwhile, the proton motive force forms a proton gradient
Oxidative phosphorylation
Dissipation of the proton gradient formed by the ETC to direct the stored potential energy into ATP synthesis
In the mitochondria,
Cytosolic content passes readily across the ___ because it has porins.
Content of the ___ is essentially contiguous with the cytosol.
The ___ is impermeable to the charged molecules; requires transporters to enter the ___.
Cytosolic content passes readily across the outer mitochondrial membrane
Content of the intermembrane space is contiguous with the cytosol
The inner mitochondrial membrane is impermeable to charged molecules; requires transporters
Pyruvate from glycolysis is transported into the mitochondrial matrix by
Active symporter with H+
(H+ going down its gradient, Pyruvate going against it)
Disrupting this dissipates the H+ gradient -> less ATP can be made
PDH complex requires what 5 vitamins?
Vitamin B1: Thiamine –> TPP (active form)
B2: Riboflavin
B3: Niacin
B5: Pantothenic acid
Lipoate/Lipoic acid
Vitamin B1 (thiamine)
Bioactivated to form TPP
Riboflavin (vitamin B2)
Used to synthesize FAD
Niacin (vitamin B3)
used to synthesize NAD
Pantothenic acid (vitamin B5)
precursor of CoA-SH
Lipoic acid (Lipoate)
Conjugated to the PDH complex via an amide bond
What is the net reaction of the PDH complex? Is it thermodynamically favorable?
Oxidative decarboxylation of pyruvate into Acetyl CoA and CO2 w/reduction of NAD+ to NADH
Yes, it is thermodynamically favorable.
As the reactions proceed, the substrate is linked to the PDH complex and channeled ___
inwards toward the center, moving from E1 > E2 > E3
(efficient substrate always stays bound to the complex)
First reaction of PDH complex - E1 is the pyruvate dehydrogenase subunit
- Pyruvate is oxidatively decarboxylated, releasing CO2
- The resulting 2C hydroxyl-ethyl group is transferred to TPP (attached to E1) –> Hydroxyethyl-TPP
The second reaction of the PDH complex - Dihydrolipoyl transacetylase (E2)
-
E1 oxidizes the hydroxyethyl group into an acetyl group
- The e- donated reduces a disulfide bond of lipoate
- E2 transfers the acetyl group from TTP to lipoyllysine (lipoate attached to a Lys on transacetylase) via thioester linkage –> reduced Acyl-lipoyllysine
The third reaction of PDH complex - Dihydrolipoyl dehydrogenase (E3)
- 3rd rxn: Acetyl group is transferred from lipoyllysineto another thioester linkage in CoA –> Acetyl CoA
- 4th rxn: E3 reoxidizes the lipollysine and transfers the e- to FAD –> FADH2
- 5th rxn: FADH2 transfers its electrons to NAD+ –> NADH
The long ____ group swings from the active site of E1 to E2 to E3, tethering the intermediates to the enzyme complex allowing substrate channeling
lipollysine
PDH complex is regulated by
-
Mainly phosphorylation
- PDH kinase inactivates
- Phosphatase activates
- Allosteric regulation
- Direct inhibits
- Acetyl-CoA & NADH
- ATP & long-chain fatty acids (LCFA)
- Directly activates - CoA, AMP, NAD+
- Direct inhibits
Regulation of PDH kinase (inactivates the PDH complex)
- Activated by: acetyl-CoA & NADH (products)
- Inactivated by:
- Pyruvate (substrate)
- ADP (indicates need ot make more ATP)
Ca++ has what impact on phosphatase?
Activates phosphatase
–> activates PDH
Beriberi
thiamine deficiency (seen in alcoholics)
-
Disrupts the PDH complex and CAC enzyme, a-KGDH
- neurological & cardiovascular disorders
- lactic acidosis
Why does beriberi cause neurological disorders
Nerves prefer to metabolize glucose.
- Disrupting PDH & a-KGDH means they can only metabolize glucose through glycolysis –> way less E
- Less acetyl-CoA for FA synthesis
- Always have a lot of glucose, so the body makes little ketone bodies (alternate source for muscle & nerves)
Treatment for beri beri
Increasing ketogenic amino acids & fat to bypass the PDH complex & generate acetyl-CoA
Arsenite or Mercury poisoning
Binds tightly to SH groups to disrupt lipollysine in PDH & a-KGDH
Cardiac & neurological problems, similar to beriberi
Leigh’s Disease
Subacute necrotizing encephalomyelopathy caused by defective PDH complex genes (usu E1)
Similar symptoms to beriberi, but rapid progressive degeneration of the CNS and presents w/ hyptonia seizures; ophthalmaloplegia (eyes don’t track together); ataxia; extreme episodes of lactic acidemia; symmetricla patches of dying brain cells
CAC / TCA cycle / Krebs Cycle
Further oxidation of carbs (from glycolysis), fats (FA B-oxidation, and many aa to generate ATP.
- Transfers e- from acetyl-CoA to NAD+ to FAD while the acetyl is oxidized to CO2
- 3 redox rxns make NADH
- 1 redox rxn makes FADH2
- NADH & FADH2 runs through the ETC and oxidative phosphorylation pathway -> ATP
- A GTP is produced by substrate level phosphorylation
2/3 of our ATP is generated by the production of __ from the CAC
NADH, FADH2, and GTP
First CAC reaction
Citrate synthase: Condenses 2-carbon acetyl-CoA with a 4-carbon oxaloacetate to form the 6-carbon citrate.
Irreversible
Feedback inhibition from citrate (which also inhibits glycolytic PFK-1)
which of the 8 reactions of CAC are redox reactions? What do they make?
Rxns 3, 4, 6, 8
Reduces 4 electron carriers to produce 3NADH and 1FADH2
In the CAC, reaction ___ is a substrate-level phosphorylation reaction, producing a molecule of __.
Rxn 5 is a substrate phosphorylation rxn making GTP
The second CAC reaction
Citrate –aconitase–> isocitrate
Isomerization rxn
The third reaction of CAC
Isocitrate + NAD+
–<strong>Isocitrate dehydrogenase</strong>-->
a-ketoglutarate + NADH2 + CO2
Irreversible
What is the 4th reaction of CAC?
a-ketoglutarate + NAD+
–aKGDH complex–>
succinyl-CoA + NADH + CO2
Generates the second NADH and the second CO2
5th reaction of CAC
Succinyl-CoA –succinyl-CoA synthetase–> Succinate + GTP
Enzyme is named after the reverse rxn; also goes by succinylthiokinase.
This is the substrate phosphorylation rxn!
6th rxn of CAC
Succinate + FAD –succinate dehydrogenase-> Fumarate + FADH2
thsi enzyme is associated with the mitochondrial membrane, unlike the rest of the CAC enzymes in the matrix; more efficient.
Final 2 reactions of CAC
Fumarate + H2O –fumarase–> malate
Malate + NAD+ –malate dehydrogenase–> oxaloacetate + NADH
Equation of CAC
Acetyl-CoA + 3NAD + FAD + GDP+Pi + 2H2O
–>
2CO2 + 3NADH + FADH2 + GTP + 2H+ + CoASH
Each NADH molecule supplies enough electron reducing power to set up a proton motive force used to synthesize ___ ATP molecules
Each FADH2 molecule can generate __ ATP
NADH - 2.5ATP
FADH2 - 1.5ATP
The total ATP form 1 round of CAC is
10 ATP
3NADH -> 7.5 ATP
FADH2 -> 1.5
GTP -> 1
The CAC is allosterically regulated within the mitochondria by..
ADP & Ca2+stimulates rxns 3 & 4
**NADH** inhibits rxns 3, 4, and 8
Citrate feedbacks & inhibits citrate synthase.
CAC intermediates are precursors for other biosynthetic pathways.
Citrate
a-ketoglutarate
Oxaloacetate
Succinyl CoA
Citrate - FA & sterols
a-Ketoglutarate -> Glutamate -> aa, purines
Oxaloacetate -> Aspartate -> aa, purines, pyrmidines
Succinyl CoA -> porphyrins, heme
Anaplerotic reactions - what are they and what’s an example?
Reactions that replenish the CAC intermediates
Ex) Pyruvate carboxylase (mitochondrial form)
Pyruvate carboxylase reaction mitochondrial vs cytosolic
Both turn pyruvate to oxaloacetate (anaplerotic)
Mitochondrial form does it for the CAC
Cytosolic form does it for gluconeogenesis
5 major anaplerotic reactions
- Pyruvate –Pyruvate carboxylase–> oxaloacetate
- Aspartate –> oxaloacetate
- aa –> Glutamate -> a-ketoglutarate
- Val, Ile, or FA –> succinyl-CoA
- aa –> Fumarate
Glycolysis produces NADH in the cytosol - how does its electrons get into the mitochondria to use ETC? how does this affect ATP production?
Glycerol phosphate shuttle - 1.5 ATP per NADH
Malate-aspartate shuttle - 2.5 ATP per NADH
Glycerol phosphate shuttle
- DHAP + NADH –<strong><em>Cytosolic G3P dehydrogenase </em></strong>–> G3P + NAD+ + H-
- G3P + FAD –<em><strong>IMM-bound G3P dehydrogenase</strong></em> –> DHAP + FADH2
- The e- from G3P oxidation are used to reduce FAD in the matrix
The reducing equivalents that were in NADH in the cytosol are now in FADH2 in the mitochondrial matrix.
Reactions of the malate-aspartate shuttle
(liver & heart)
- malate + NAD+ –<em><strong>malate dehydrogenase</strong></em>–> oxaloacetate + NADH
- Transamination rxn: a-amino group from Glu is transferred to Oxaloacetate and replaced wiht a keto group.
- –> aketoglutarate
- –> Aspartate
- Requires PLP as a cofactor
- Enzyme is Aspartate transaminase
AST reaction
convert Aspartate to Oxaloacetate
convert a-ketoglutarate to Glutamate
used in liver fxn tests
Malate-Aspartate shuttle
Getting the electrons of NADH from the cytosol into the mitochondria
Why does NADH form 2.5 ATP and not 3?
Glutamate protonated in the cytosol to glutamic acid.
Once in the matrix, glutamic acid turns back into glutamate, causing a loss of proton motive force across the IMM
Which steps of CAC include carbon dioxide as a product?
Rxns 3 & 4
What CAC enzyme is subject to product inhibition?
citrate synthase is inhibited by citrate
What is the energy yield from glucose in the presence of oxygen but absence of PDH?
2 ATP from each mole of glucose in glycolysis
+
ATP from running the 2NADHs in the cytosol thru ETC and OxPhos (1.5 or 2.5 depending on the shuttle).
-> 5ATP (2 + 3) if glycerol phosophate shuttle
-> 7ATP (2 + 5) if malate aspartate shuttle
In brain and neural tissue, more PDH complexes are
unphosphorylated (active)
Energy charge (adenylate charge)
