Krebs cycle Flashcards
link reaction 1 -
what happens and what enzymes/coenzymes are used?
pyruvate is decarboxylated to hydroxyethyl-TPP by enzyme pyruvate dehydrogenase using prosthetic group TPP (thiamine pyrophosphate).
link reaction 2 -
what happens, including enzymes/coenzymes used?
two acetyl transfers by enzyme dihydrolipoyl transacetylase.
first uses coenzyme lipoamide, which oxidises the TPP to an acetyl fragment, forming acetyl dihydrolipoamide and regenerating TPP.
second uses CoA - the acetyl group is transferred to CoA, giving acetyl-CoA and dihydrolipoamide. this involves the energetically favourable hydrolysis of a thioester linkage.
link reaction 3 -
what happens, what enzymes and coenzymes are used?
dihydrolipoyl dehydrogenase uses coenzyme FAD to oxidise the dihydrolipoamide, regenerating the lipoamide anf forming FADH2.
FADH2 reduces NAD+ to form NADH, so the link reaction produces (per glucose = 2 pyruvate) 2NADH, 2CO2, 2 acetyl-CoA.
Krebs cycle -
what is the first step?
what enzyme is used?
what drives this reaction?
oxaloacetate + acetyl-CoA = citrate
citrate synthase (deprotonates methyl group on acetyl-CoA Which then acts as a nucleophile and attacks the oxaloacetate carbonyl group)
an S-citryl-CoA intermediate that is formed has an energetically favourable hydrolysis.
Krebs cycle -
what is the second step?
enzyme used?
why is this step necessary?
isomerisation of citrate to isocitrate
aconitase (just removes water and adds it back elsewhere)
conversion from 2 alcohol to 3 alcohol means in the next step the bond being broken is C-H, not C-C.
Krebs cycle -
what is step 3?
enzyme used?
what exactly does this step do?
isocitrate decarboxylated to a-ketoglutarate, producing CO2 and NADH
isocitrate dehydrogenase
main importance is the production of NADH - oxalosuccinate intermediate is used to convert a hydroxyl to a carbonyl, providing a hydride to reduce NAD+
Krebs cycle -
what happens in step 4?
enzyme used?
how is coupling used?
a-ketoglutarate is decarboxylated, producing CO2, and oxidised, producing NADH and succinyl CoA.
a-ketoglutarate dehydrogenase
the oxidation (-ve delta G) is coupled to formation of NADH
Krebs cycle -
what is step 5?
what enzyme is used?
succinyl -CoA to succinate
Succinyl-CoA synthetase
how does succinyl-CoA synthetase work?
a phosphate group acts as a nucleophile and replaces CoA to form a succinyl phosphate intermediate
a histidine side chain acts as a nucleophile, removing the phosphate to form succinate and phosphohistidine
the phosphate is then transferred to ADP forming ATP via SLP
how are these phosphate transfers able to occur in the succinyl-CoA synthetase mechanism able to occur?
due to a high phosphoryl transfer potential
Krebs cycle -
what is step 6?
what enzyme is used?
what is made?
succinate converted to fumarate
succinate dehydrogenase
FAD is used, reduced to FADH2
why is FAD used and not NAD+?
formation of the C=C bond does not provide enough free energy to reduce NAD+, unlike C=O
Krebs cycle -
Step 7?
Enzyme used?
What is this step used for?
fumarate to malate
fumarase adds water across the C=C double bond
this is the only way to further oxidise the carbon without forming the difficult carbon carbon triple bond, so we facilitate oxidation in the next step instead.
Krebs cycle -
Step 8?
enzyme used?
malate to oxaloacetate, oxidise malate to reduce NAD+ to NADH
malate dehydrogenase
total products from the Krebs cycle from x2 acetyl-CoA?
NADH from step 3,4,8, so 6NADH in total
C02 in step 4 so 2 CO2 all together
ATP in step 5 so 2 ATP
FADH2 in step 6 so 2 FADH2
why is the Krebs cycle so complicated?
seems a bit much all to oxidise acetate, a 2 carbon fragment.
it require the breaking of C-C bonds, which can only occur between 2 carbons adjacent to a carbonyl group, or cleavage of a hydroxyketone
2nd option would require hydroxylation, which isn’t energetically favourable so forming a citrate provides the cleavage site explained in option 1
the Krebs cycle is amphibolic - what does this mean?
its intermediates are syphoned off to be start points in other biochemical pathways, e.g. oxaloacetate is used in gluconeogenesis
how did Krebs discover the cycle?
used minced pigeon muscle as this has lots of mitochondria.
added malate, succinate, citrate, fumarate.
resulted in massive oxygen uptake, way more than was needed to oxidise these acids alone.
so he concluded addition of these acids must work catalytically to oxidise an endogenous substance - acetate.
how did Krebs utilise poisons?
added malonate - inhibited respiration and caused a build up of succinate - acts as a competitive inhibitor of succinate dehydrogenase, so this enzyme must play a key role in respiration.
how did Krebs know it was a cycle?
no matter what acid he added, malonate always ended up giving the same result, so the process must be a cycle, unable to move on to the next step if one is inhibited
why is it improtant that the three enzymes in the pyruvate dehydrogenase complex be near eachother?
to prevent toxic substances from being produced in unwanted side reactions as each substrate diffuses straight from one enzyme to the other
what are the three enzyme (and their coenzymes) in the pyruvate dehydrogenase complex?
E1 = pyruvate dehydrogenase (funnily enough it decarboxylates actually) and TPP
E2 = dihydrolipoamide transacetylase, lipoamide and Acetyl-CoA
E3 = dihydrolipoyl dehydrogenase, FAD (does make FADH2 but only temporarily as this reduces an NAD+ to form NADH)
what provides oxygen atoms to make CO2 from acetyl-CoA?
H2O, not oxygen
Oh, what are you doing right now?
that’s right. slaying
