5. Citric acid cycle Flashcards
Consumption of oxygen (respiration) depends on the rate of ________ and ________ reactions.
cytosol
A 1930 study of oxidation of glucose in muscle showed what?
Malonate inhibiting respiration
1935: Szent-Gyorgyi: demonstrated that little amounts (catalytic amounts) of succinate, fumerate, malate or oxaloacetate acelerated the rate of ___________.
respiration
He believed these must be acting as catalysts. But they aren’t enzymes. How can they act as catalysts?
participates but then comes back in tact after doing the reaction.
How many reactions in TCA cycle?
8
In order, name the reactions of TCA cycle by naming their enzymes. Add a * to the oxidation steps.
- Citrate synthase
and reaction:
CoA-SH, NAD, TPP, lipoate, FAD
Why is PDC exergonic?
exergonic because it is an oxidation reaction
Advantages of multienzyme complex:
1.Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion.
Name the 3 enzymes part of the PDC multi-enzyme complex:
E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor bound to E1
What are the three forms of lipoamide we see and what enzyme is it usually attached to?
Oxidized (disulfide bond)
What are the 5 co-factors found in PDC mechanism?
TPP, lipoamide, co-A, FAD, and NAD+
The first half of PDC mechanism is very similar to what?
First half is very similar to pyruvate decarboxylation reaction of alcohol fermentation
Provide the steps to the mechanism of PDC:
- TPP (with E1 bound to it) carbanion nucleophilicaly attacks carbonyl carbon of pyruvate, lose the double bonded O to form OH
Regulation of PDC by phosphorylation:
When PD kinase phosphorylates PDC to make it inactive
What activates and inhibits PD kinase?
PD kinase is activated by NADH and acetyl coa (makes sense because PDC forms NADH and acetyl coa)
What activates PD phosphatase?
PD phosphatase is activated by Mg2+ and Ca2+
How can hormones regulate PDC?
Insulin activates insulin-stim kinase which activates PP1, which activates PDC to form more acetylcoA
Why is insulin regulation of PDC somewhat contradictory?
Insulin is an anabolic hormone. However PDC catabolizes pyruvate, so it is a catabolic enzyme.
What is the cause of Beriberi?
If TPP is not there, PDC is not working. This results in issues with the brain, which depends on glucose. Brain cannot use anaerobic mechanism. (PDC is the entry-point for oxidative glucose catabolism. Brain has no fermentation to regenerate NAD+, so glycolysis cant just be used. U need OXIDATIVE catabolism.) So no TPP = no PDC = no oxidative glucose catabolism = no brain fuel = neurological symptoms
Why are arsenic compounds poisonous?
the sulfhydryl groups of dihydrolipoamide are covalently inactivated by arsenic compounds
Arsenic Compound poisoning: Inactivation of _______ of PDC, and other proteins
E2
Name and describe reaction 1 of CAC:
Citrate synthase: Formation of Citroyl CoA intermediate. Binding of Oxaloacetate to the enzyme results in conformational change which facilitates the binding of the next substrate, the acetyl Coenzyme A. There is a further conformational change which leads to formation of products.
Provide reaction 1 of CAC:
Acetyl-CoA + Oxaloacetate + H20 –> citrate (by citrate synthase)
describe the free energy of reaction 1 of CAC and what the mechanism is:
VERY exergonic
Describe structure of citrate:
Tricarboxylic acid with one of the carbons having an OH
Name and describe reaction 2 of CAC:
Aconitase: This enzyme catalyses the isomerization reaction by removing and then adding back the water ( H and OH ) to cis-aconitate in at different positions. Isocitrate is consumed rapidly by the next step thus deriving the reaction in forward direction.
Isocitrate is formed by removing water to form double bond and then adding water so there is addition of OH to C3 and H to C2 (switch places). True or false?
TRUE
The formation of isocitrate is an _____________ attack on a symmetric structure. Why?
Asymmetric
Name and describe reaction 3 of CAC:
Isocitrate dehydrogenase: converts isocitrate to a-ketoglutarate + CO2
Name and describe reaction 4 of CAC:
- a-Ketoglutarate dehydrogenase complex: a-ketoglutarate to succinyl coA
True or false?
TRUE
So what are the co-factors of a-ketoglutarate dehydrogenase complex?
TPP, FAD, NAD+, lipoamide, CoA are the co-factors here too
Name and describe reaction 5 of CAC:
Succinyl CoA synthetase: Succinyl-CoA is converted to Succinate, forming GTP in the process and releasing CoA
Formation of GTP in Succinyl CoA synthetase reaction:
Succinyl co-A binds to succinyl co-A synthetase and Pi binds to the succinyl structure to replace coA
Name and describe reaction 6 of CAC:
Succinate Dehydrogenase:
How can succinate dehydrogenase be inhibited?
competitively by malonate (Similar structure to succinate)
Get info from slide 27 question
okay
Name and describe reaction 7 of CAC:
Fumarase: Hydration of Fumarate to L-malate: It is a highly stereospecific enzyme. Cis-Maleate (the cis form of fumarate) is not recognized by this enzyme.
Name and describe reaction 8 of CAC:
L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It is an NAD+dependent enzyme. Reaction is pulled in forward direction by the next reaction (citrate synthase reaction) as the oxaloacetate is depleted at a very fast rate.
So what are the standard and actual delta G signs of reaction 8?
Standard = +
The two carbon acetyl group generated in PDC reaction enter the CAC, and two molecules of ________ are released in one cycle. Thus there is complete ________ of two carbons during one cycle. Although the two carbons which enter the cycle become the part of oxaloacetate, and are released as CO2 only in the ________ round of the cycle. The energy released due to this oxidation is conserved in the reduction of 3 ________ , 1 ________ molecule and synthesis of one ________ molecule which is converted to ________ .
CO2
How many ATP per NADH and FADH2?
2.5, 1.5
Indicate how much total ATP from glycolysis, PDC and CAC is received and detail where it comes from
From pyruvate in CAC, we get 4 NADH (1 pdc and 3 from cac), 1 FADH2, and 1 Atp from gtp. So PDC and CAC makes 12.5 ATP from one pyruvate. We get 2 pyruvates from glucose, so 25 ATP from one glucose from pyruvate onwards.
Anaerobic bacteria use an incomplete form of the CAC. What is missing and why do they run this?
They do not have a-ketoglutarate dehydrogenase. They use this incomplete CAC for the production of biosynthetic products (AAs, nucleotides, heme, etc.)
How did farmers take advantage of anaerobic bacteria ability to use the incomplete cycle?
Idea was to go to crops that were full of aluminum to chelate it with citrate produced by anaerobic bacteria to allow crops to grow by adding sugar.
The citric acid cycle is considered to have a ____________ nature
amphibolic
Explain the amphibolic nature of the CAC:
This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis.
read through amphibolic diagram on slide 33
okay
What cycle is found in plants that has CAC intermediates?
Glyoxylate cycle
Describe the glyoxylate cycle:
Start is same as CAC to form isocitrate, then you break it up by isocitrate lyase to form glyoxylate and 4C succinate is removed
Why is the glyoxylate cycle very important to plants? Explain the importance to things like oil seeds:
Allows for the formation of biosynthetic products by forming succinate, while also not making any CO2.
Ultimately, the glyoxysome allows for what to happen?
Convert FA to acetyl coa, then make carbs and proteins
Rate controlling enzymes of PDC and CAC:
- PDC
Regulation of activity of CAC by: (3)
- Substrate availability
Read through slide 37 for what specifically inhibits and activates the 4 enzymes of PDC and CAC regulation.
okay
Which reactions of glycolysis, PDC, and CAC produce CO2?
NONE during glycolysis.
Carbons have been oxidized to CO2. Completely oxidized without oxygen. Where did the oxygen come from?
The water addition in GAPDH reaction in glycolysis, citrate synthase reaction, and fumarase reaction all contribute the oxygen for oxidation.
