Lecture 2 Flashcards
What molecule is at a central point in metabolism?
What can it be converted into, and how?
Pyruvate.
Lactate - Reduction.
Acetyl CoA - Oxidative Decarboxylation.
Oxaloacetate - Carboxylation.
Alanine - Transamination.
What is pyruvate synthesised from, and by which process?
Glucose, glycolysis.
Where is Acetyl CoA produced? Is it reversible?
Mitochondria.
Not reversible.
What are the reactants involved in producing Acetyl CoA?
Pyruvate + CoA-SH.
Which enzyme and which coenzyme is used to produce Acetyl CoA?
Pyruvate Dehydrogenase (PDH), NAD+ -> NADH.
What is the MW of Pyruvate Dehydrogenase?
5-10X10^6 Da.
How many separate enzyme proteins does PDH contain? What are they?
3: E1, E2, E3.
Where is PDH found in large quantities?
Mitochondria of plants and animals.
How many cofactors does PDH require? What type of molecule are most of these? How does this relate to lethargy?
- Mostly human vitamins.
Lethargy arises from lack of these vitamins.
What is E1’s cofactor? What’s its association with the enzyme? Is the cofactor sourced from a vitamin?
Thiamine pyrophosphate. Bound to enzyme. Cofactor sourced from Thiamine (Vitamin B1).
What are E2’s cofactors? What are their associations with the enzyme? Are these cofactors sourced from a vitamin?
Coenzyme A. Free association. Cofactor sourced from Pantothenic Acid (Vitamin B5).
Lipoamide (lipoic acid + lysine residue). Bound to enzyme. Cofactor not sourced from vitamin - non-dietary.
What are E3’s cofactors? What are their associations with the enzyme? Are these cofactors sourced from a vitamin?
FAD. Bound to enzyme. Cofactor sourced from Riboflavin (Vitamin B2).
NAD+. Free association. Cofactor sourced from Niacin (Vitamin B3).
What’s very reactive on TPP (Thiamine pyrophosphate)? How does this enable it to react with carbonyl group on pyruvate?
The C between the N and S is very reactive. Causes H to readily dissociate as H+, leaving a -ve C ion that can react with carbonyl group of pyruvate.
Why (structurally) does lipoic acid attach to a lysine molecule in protein, forming lipoamine?
To form a long extendable, moveable arm.
How does the ring open up in lipoic acid?
Two sulphur atoms on it can be reduced during transfer of acetyl group, causing ring to open up,
How large is the long moveable arm in lipoamide?
14 Å.
What is E1?
How many subunits is it comprised of?
Pyruvate dehydrogenase component.
24 subunits.
What is E2?
How many subunits is it comprised of?
Transacetylase core.
8 subunits.
What is E3?
How many subunits is it comprised of?
Dihydrolipoyl dehydrogenase.
12 subunits.
How many distinct domains are there in an E2 subunit? Which component do they interact with?
3 distinct domains in one E2 subunit.
Interact with E3 component.
What 2 key reactions happen with E1 in pyruvate dehydrogenase?
Decarboxylation of Pyruvate.
Transfer of hydroxyethyl group to lipoamide (transacetylation).
How does the decarboxylation of pyruvate occur?
TPP carbanion spontaneously forms, producing TPP and H+.
Carbanion then adds to carbonyl group of pyruvate, forming hydroxyethyl-TPP, losing CO2 and reacting with H+ in process.
How does transacetylation occur in E1?
Hydroxyethyl group from hydroxyethyl TPP oxidised to form acetyl group. Reacts with lipoamide to form carbanion of TPP and acetyllipoamide.
How is Acetyl CoA formed in E2?
Acetyl group from acetyllipoamide transferred to CoA, forming Acetyl CoA and dihydrolipoamide.
How is FAD reduced in E3 of PDH?
By reaction with Dihydrolipoamide to form Lipoamide and FADH2.
I.e. Regeneration of lipoamide.
How is FAD then reoxidised after reduction via dihydrolipoamide?
Reacts with NAD+ transferring one hydrogen and offloading the other, forming NADH + FAD + H+.
What does the structural integration of three kinds of enzymes in PDH enable?
The co-ordinated catalysis of a complex.
What 3 advantages do the proximity of the enzyme subunits provide in PDH?
Reduces the number of side reactions.
Maximises efficiency.
Maximises rate of reaction.
Why is it important that all organisms can control the amount of pyruvate converted into Acetyl CoA? What happens if the concentration is too high?
Because it’s an irreversible step, and a commitment to energy production.
High concentrations of reaction products inhibit PDH activity (NADH, Acetyl CoA, ATP).
What 3 cofactors can activate the PDH complex?
NAD, ADP AMP.
What type of reaction is the primary regulator of the PDH complex?
What enzyme performs most of the regulation, and how does it work?
Phosphorylation.
PDH kinase, can phosphorylate at 3 serine resides on the E1 subunit, causing loss of enzyme activity or deactivation.
When deactivated, what stimulates PDH kinase activity?
NADH, ATP, Acetyl CoA.
When activated, what inhibits PDH kinase activity?
NAD+, ADP/AMP, CoA.
What stimulates PDH phosphatase? What does it activate, and where is this important?
Elevation of cytosolic Ca2+.
Activates PDH - important in muscles.
What enzyme inactivates PDH by phosphorylation?
PDH kinase.
What enzyme activates PDH by dephosphorylation?
PDH phosphatase.
Beriberi comes from which language?
Sinhalese. :D
What causes beriberi?
Deficiency of thiamine (vitamin B1).
Where is beriberi a serious health problems and why?
Far East, as polished rice usually consumed, which removes primary source of thiamine.
What common condition does beriberi sometimes manifest itself in?
Alcoholism.
What type of disorder is beriberi, and why?
Neurological, because glucose is the primary source of energy for the CNS, as fats can’t be used.
What are you likely to find high levels of in the blood of a patient with beriberi?
Pyruvate and lactic acid.
What type of poisoning has similar symptoms to beriberi? Why?
Mercury and arsenite, as arsenite can react with dihydrolipoamide from E2 of PDH, producing an arsenite chelate on the enzyme.
How does BAL (British anti-lewisite) cure arsenite poisoning?
Binds arsenite chelate on enzyme, and is then excreted, restoring normal enzyme.
