ATP: acetylcoA generation and utilization, mitochondrial electron transport Flashcards
What is the general function of PDH?
To convert pyruvate to acetyl coA
Describe the PDH complex.
It is made of 3 enzymes: E1 (decarboxylase), E2 (dihydrolipoyl transacetylase), E3 (dihidrolipoyl dehydrogenase)
Describe the prosthetic groups of PDH. What do they do?
- E1 has thiamine pyrophosphate. The thiamine acidic proton leaves, leaving a carbanion that attacks and decarboxylates pyruvate, resulting in 2-carbon intermediate bound to TPP.
- E2 has lipoamide with oxidized thiol which is reduced by transfer of two-carbon intermediate from TPP. Lipoamide is tethered to E2 via a long lysine arm, allowing it to move between E1 and E3 prosthetic groups. (there are two lipoamide arms to help with reaching)
- E3 has FAD prosthetic group which re-oxidizes lipoamide thiol, as the two-carbon intermediate bound to lipoamide is released and bound to coA. FADH2 is then re-oxidzed by NAD.
Difference in electron carrying capacity of FAD and NAD?
NADH carries two electrons on one hydride. FADH2 carries two electrons, each on one hydrogen.
What are the catalytic cofactors of PDH? Stoichiometric cofactors?
catalytic: TPP, lipoamide, FAD
stoichiometric: coA, NAD
What is the benefit of catalysis by multiple enzymes in the PDH complex?
- increases reaction rate
- minimizes side reactions
In general, how does PDH accomplish its chain of coordinated reactions?
PDH has many subunits in a highly ordered assembly.
Describe the model of PDH subunit organization and why it makes sense for what PDH must accomplish.
- E1 is on the outside of the complex because it must accept pyruvate
- E2 is on the inside of the complex along with E3, because E2 must move its lipoamide arm back and forth between E1 and E2, so must be able to reach both.
Describe the regulation of PDH complex.
- NADH and acetyl coA competitively inhibit E3 and E2, respectively
- covalent regulation by phosphorylation: E1 is inhibited by phosphorylation (kinases that do this are activated by ATP, NADH, and acetyl coA)
- calcium and insulin activate the phosphatase that removes phosphate from PDH and re-activates it
How is PDH transcriptionally regulated during starvation?
Under starvation, body wants to preserve glucose for the brain to do glycolysis, so does not break it down beyond pyruvate.
- transcription of PDH kinase increases to inactivate E1
- txn of PDH phosphatase decreases
Describe in general the carbon chain lengths iin TCA intermediates.
2C pyruvate + 4C OXA -> 6C citrate
carbons are lost one at a time via decarboxylation reactions to recycle back to 4C OXA
Describe, per pyruvate molecule, the products made in the TCA cycle.
- 3 NADH made
- 1 FADH2 made
- 1 GTP made
Describe the initiating reaction of the TCA.
citrate synthase catalyzes condensation of acetyl coA with OXA to form citrate. Reaction is irreversible by the high energy thioester bond of acetyl coA, making this the committed step of the TCA
Describe the TCA reaction catalyzed by aconitase.
Aconitase removes H2O from citrate to form cis-aconitate, and then adds it back on to cis-aconitate in a different position to form isocitrate. This preps isocitrate for the first decarboxylation reaction (and first reduction of NAD)
How does OXA help control citrate synthase activity?
The binding of OXA, considered a catalyst, to citrate synthase induces a conformational change that provides the binding site for acetyl-coA. This prevents the loss of the high-energy thioester bond to hydrolysis.
Which enzyme of the TCA is analagous to PDH?
a-ketogluterate dehydrogenase. It too leads to the formation of CO2 and NADH, as well as succinyl coA (high energy thioester formed) by feeding in coA and NAD. The new high energy bond is used by succinyl thiokinase to perform substrate-level phosphorylation of GDP (succinyl phosphate intermediate).
