Entering the TCA cycle Flashcards
What role does the TCA cycle play?
A central role.
Its a cycle.
Its highly interconnected
What can enter the TCA cycle as Acetyl CoA?
Fats: fatty acids and glycerol
Polysaccharides: glucose and other sugars
Proteins: amino acids
Importance of the TCA cycle (3 points)
- Its central to aerobic metabolism and ATP production, generating the bulk of NADH and FADH2, which is going to be oxidized later to generate ATP
- Links the oxidation of metabolic fuels to ATP synthesis (fatty acids, amino acids, carbohydrates can be oxidized to Acetyl coA)
- Provides metabolic intermediates for several biosyntehtic pathways (thats why you need to run the TCA cycle even if you dont need energy)
Coenzyme A is the activated carrier of
Acetyl groups (2C)
CoEnzyme A is also known as
CoASH
What are the 2 substrates needed to start the TCA cycle
- Oxaloacetate (4C) generated by pyruvate carboxylase. will be regenerated after 1 run of the cycle
- Acetyl CoA (2C), generated from pyruvate dehydrogenase complex (PDH), fatty acid degradation, amino acid breakdown.
-> Pyruvate is used to syntheisze Acetyl coA and OAA.
Pyruvate needs to be transported from the cytosol (glycolysis/gluconeogenesis) into the mitochondrial matix
How is pyruvate transported inside the mitochondria? What are the 2 fates of pyruvate after this
Transported via mitochondrial pyruvate carrier (MPC).
2 fates:
Inside the mitochondria, pyruvate can be used by pyruvate carboxylase -> OAA and pyruvate dehydrogenase -> Acetyl coA
How would a 100% lipids diet affect the TCA cycle?
Acetyl coA can be gained from amino acids, fatty acids, glyrols, carbohydrates. (oxidation of these molecules)
But you still need OAA, which comes from pyruvate -> need carbs
What will be the substrates of TCA, starting with Glucose
Glucose -> pyruvate -> 2 ways into Acetyl coA or Oxaloacetate
What will be the substrates of TCA, starting with fatty acids
fatty acids -> acetyl coA.
Pyruvate -> OAA
cycle stats
What is the pyruvate dehydrogenase complex?
A mitochondrial matrix enzyme, which OXIDATIVELY DECARBOXYLATES pyruvate to form acetyl coA
What is PDH made of
3 enzymes: 2 dehydrogenases and 1 transcetylase
Being part of a complex facilitates substrate channeling. What does substrate channeling do.
It allows substrates to travel from 1 active site to another with the help of the lipoamide arm (LD)
What is the overall reaction of the PHD? Is this irreversible?
irreversible reaction: Pyruvate + CoA + NAD+ -> Acetyl CoA + Co2 + NADH + H+
What are cofactors?
Some enzymes require the addition of another non-protein molecule to function as an enzyme
What are the cofactors of PDH?
LD: lipoamide domain
TPP: thiamine pyrophosphate
FAD: flavin adenine dinucleotide
Explain the 3 reactions of PDH
- E1. Decarboxylation of pyruvate, carried out by pyruvate dehydrogenase
TPP acts as cofactor to extract the Co2
Lipoamide arm carries the 1st hydrogen
- Transfer actyl group to coA with the help of cofactor: lipamide arm
enzyme: dihydrolipoyl transecetylase
lipoamide arm carries 2nd hydrogen
- Regeneration of oxidized lipoamide with the help of cofactor FAD.
like it loads electrons/protons onto FADH2 -> reduced to NADH
-> oxidized to NAD+
Generates NADH
Enzyme: dihydrolipoyl dehydrogenase
What does the PDH complex look like
E2 in the center with lioamide arms, carry substrate from active site to active site.
Increases overall reaction rate and minimizes side reactions
Purpose of enzyme cofactors/coenzymes
Expand the repertoire of reactions
Purpose of cofactors in PDH
TPP: extract Co2 from pyruvate
Lipoamide: hold tightly to acetyle groups and transfer them
FAD: restore lipoamide
Humans cannot make these cofactors -> need to take vitamins
Why must steps in PDH be coupled
Steps must be coupled to preserve the free energy derived from the decarboxylation to drive NADH and acetyl CoA formation
What is the rate limiitng step of the PDH
(E1) Pyruvate dehydrogenase and TPP:
decarboxylation of pyruvate
Step by step sequence of reactions of PDH.
- Pyruvate is decarboxylated to from hydroxyethyl-TPP by E1, releasing Co2 as the first product.
Enzyme: pyruvate decarboxylase
- The lipoamide arm of E2 moves into the active site of E1
- E1 catalyzes the transfer of the 2 carbon group to the lipoamide group, forming the acetyl-lipoamide complex, enters E2.
Lipoamide arm is reduced: 1 SH
- E2 catalyzes the transfer of the acetyl moiety to form product Acetyl- CoA. the lipoamide arm then swings to active site of E3.
ENzyme: dihydroli transetelase
- E3 catalyzes the oxidation of the lipoamide arm by FAD
Enzyme: dihydroli dehydrogenase
- The final product NADH is produced by the reoxidation of FADH2
is the conversion of pyruvate to Acetyl CoA irreversible
yes. once pyrvuate is turned into acetyl CoA, can no longer be converted into glucose (in humans)
What fates does Acetyl coA have? How is it regulated
Acetyl coA has 2 main fates:
1. Metabolism by the citric acid cycle
2. incorporation into fatty acids
Regulated by 2 mechanisms.
1. Allosteric regulators
2. covalent modifications (phosphorylation)
Is feedback inhibition a form of allosteric regulation?
Yes. The end product binding to the allosteric site delays or prevents the enzyme’s activity, resulting in slight or no further end product being produced.
How is PDH regulated allosterically? (-) and (+)
(-) ATP
Acetyl coA, NADH: feedback inhibtion
Increase in Acetyl CoA -> inhibits E2 (transacetulase) -> no need to metabolize pyruvate to acetyl coA.
Increase in NADH -> inhbits E3 (dehydrogenase) -> no need to metabolize pyruvate to acetyl coA
(+) Pyruvate: more substrate -> make more
How is PDh regulated by covalent modication
E1 of PDH can be phosphorylated -> inactivates E1
Not phosphorylated -> remains active
PDH kinase is active -> phosphorylates E1 -> inactivates E1
PDH phosphatase is active -> dephosphorylates E1 -> activates E1
How is the phosphorylation of E1 regulated
For the kinase:
(+) ATP, NADH
-> phosphrylates E1
-> promotes the activity of kinase -> PDH is inactive -> cells have enough energy
(-) ADP, pyruvate
-> inhibits phosphrylation of E1
-> decreases activity of kinase -> E1 is dephsophrylated -> PDH is active
For the phosphatase
Insulin -> promotes activity of phosphatase -> dephosphorylate -> makes PDH active -> cause there glucose (espcially after eating), you should break it down.
(+) Calcium
muscles exert energy -> calcium increases -> stimulates the dephosphorylation -> PDH active -> obtain energy from TCa cycle
Insulin activates/deactivates phosphatase
Activates
Effect of insulin on the synthesis of Acetyl CoA
Insulin promotes the synthesis of Acetyl CoA
Regulators of PDH, PDH kinase (PDH off), PDH phosphatase)
PDH:
(-) ATP, NADH
(+) Pyruvate
PDH kinase:
(+) ATP, NADH
(-) ADP, Pyruvate
PDH phosphatase:
(+) Ca2+, insulin
Ca2+ stimulates phosphatase/kinase
Phosphatase
When you are at rest, that state is referred to as, when you are exercising, that state is referred to as:
at rest: high energy
during exerise: low energy
When you are at rest, what ratios are high? What affects the PDH
High ATP/ADP, NADH/NAD, Acetyl CoA/CoA
ATP (-) E1 (because it promotes kiinase phosphorylation -> inactivates PDH
Acetyl coA (-) E2
NADH (-) E3 (feedback inhibition)
-> no need to run TCA cycle
When you are exercising, what ratios are low? What affects the PDH?
Low NADH/NAD+, Acetyl CoA/CoA, ATP/ADP
ADP -> reduces kinase activity ->
kinase does not phosphorylate PDH -> activates PDH (E1)
Calcium stimulates phosphatase. Removes phosphate group from PDH. PDH is on when energy charge is low.
Pyruvate (+) PDH
-> need to run TCA to obtain energy
How does PDH link glycolysis and the TCA cycle?
By forming acetyl coA from pyruvate
The fates of pyruvate and reciprocal regulation
Acetyl CoA controls both pyruvate carboxylase and pyruvate dehydrogenase
Acetyl coA feedback inhibits pyruvate dehyogenase
Acetyl coA (+) pyruvate carboxylase (indicates more OAA is needed to start TCA)
Pyruvate is an intermediate for glucose oxdiation and precursor for..
the synthesis of glucose, glycerol, fatty acids and amino acisd
Explain the increase in pyruvate and lactate, defect in muscle activity when PDH is affected
lack of PDH
-> build up of pyruvate
-> build up of lactate since pyruvate undergoes lactic fermentatin
-> lack of energy -> muscles affectsed
Treatmnets for PDH deficiency
keto diet
In diabetic patients, there is an increase/decrease in PDH kinase activity
increase -> target the inhibition of kinase
