5. Citric acid cycle

Question 1

Consumption of oxygen (respiration) depends on the rate of ________ and ________ reactions.

Answer 1

cytosol

Question 2

A 1930 study of oxidation of glucose in muscle showed what?

Answer 2

Malonate inhibiting respiration

Question 3

1935: Szent-Gyorgyi: demonstrated that little amounts (catalytic amounts) of succinate, fumerate, malate or oxaloacetate acelerated the rate of ___________.

Answer 3

respiration

Question 4

He believed these must be acting as catalysts. But they aren’t enzymes. How can they act as catalysts?

Answer 4

participates but then comes back in tact after doing the reaction.

Question 5

How many reactions in TCA cycle?

Answer 5

8

Question 6

In order, name the reactions of TCA cycle by naming their enzymes. Add a * to the oxidation steps.

Answer 6

1. Citrate synthase

Question 7

and reaction:

Answer 7

CoA-SH, NAD, TPP, lipoate, FAD

Question 8

Why is PDC exergonic?

Answer 8

exergonic because it is an oxidation reaction

Question 9

Advantages of multienzyme complex:

Answer 9

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.

Question 10

Name the 3 enzymes part of the PDC multi-enzyme complex:

Answer 10

E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor bound to E1

Question 11

What are the three forms of lipoamide we see and what enzyme is it usually attached to?

Answer 11

Oxidized (disulfide bond)

Question 12

What are the 5 co-factors found in PDC mechanism?

Answer 12

TPP, lipoamide, co-A, FAD, and NAD+

Question 13

The first half of PDC mechanism is very similar to what?

Answer 13

First half is very similar to pyruvate decarboxylation reaction of alcohol fermentation

Question 14

Provide the steps to the mechanism of PDC:

Answer 14

1. TPP (with E1 bound to it) carbanion nucleophilicaly attacks carbonyl carbon of pyruvate, lose the double bonded O to form OH

Question 15

Regulation of PDC by phosphorylation:

Answer 15

When PD kinase phosphorylates PDC to make it inactive

Question 16

What activates and inhibits PD kinase?

Answer 16

PD kinase is activated by NADH and acetyl coa (makes sense because PDC forms NADH and acetyl coa)

Question 17

What activates PD phosphatase?

Answer 17

PD phosphatase is activated by Mg2+ and Ca2+

Question 18

How can hormones regulate PDC?

Answer 18

Insulin activates insulin-stim kinase which activates PP1, which activates PDC to form more acetylcoA

Question 19

Why is insulin regulation of PDC somewhat contradictory?

Answer 19

Insulin is an anabolic hormone. However PDC catabolizes pyruvate, so it is a catabolic enzyme.

Question 20

What is the cause of Beriberi?

Answer 20

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

Question 21

Why are arsenic compounds poisonous?

Answer 21

the sulfhydryl groups of dihydrolipoamide are covalently inactivated by arsenic compounds

Question 22

Arsenic Compound poisoning: Inactivation of _______ of PDC, and other proteins

Answer 22

E2

Question 23

Name and describe reaction 1 of CAC:

Answer 23

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.

Question 24

Provide reaction 1 of CAC:

Answer 24

Acetyl-CoA + Oxaloacetate + H20 –> citrate (by citrate synthase)

Question 25

describe the free energy of reaction 1 of CAC and what the mechanism is:

Answer 25

VERY exergonic

Question 26

Describe structure of citrate:

Answer 26

Tricarboxylic acid with one of the carbons having an OH

Question 27

Name and describe reaction 2 of CAC:

Answer 27

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.

Question 28

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?

Answer 28

TRUE

Question 29

The formation of isocitrate is an _____________ attack on a symmetric structure. Why?

Answer 29

Asymmetric

Question 30

Name and describe reaction 3 of CAC:

Answer 30

Isocitrate dehydrogenase: converts isocitrate to a-ketoglutarate + CO2

Question 31

Name and describe reaction 4 of CAC:

Answer 31

4. a-Ketoglutarate dehydrogenase complex: a-ketoglutarate to succinyl coA

Question 32

True or false?

Answer 32

TRUE

Question 33

So what are the co-factors of a-ketoglutarate dehydrogenase complex?

Answer 33

TPP, FAD, NAD+, lipoamide, CoA are the co-factors here too

Question 34

Name and describe reaction 5 of CAC:

Answer 34

Succinyl CoA synthetase: Succinyl-CoA is converted to Succinate, forming GTP in the process and releasing CoA

Question 35

Formation of GTP in Succinyl CoA synthetase reaction:

Answer 35

Succinyl co-A binds to succinyl co-A synthetase and Pi binds to the succinyl structure to replace coA

Question 36

Name and describe reaction 6 of CAC:

Answer 36

Succinate Dehydrogenase:

Question 37

How can succinate dehydrogenase be inhibited?

Answer 37

competitively by malonate (Similar structure to succinate)

Question 38

Get info from slide 27 question

Answer 38

okay

Question 39

Name and describe reaction 7 of CAC:

Answer 39

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.

Question 40

Name and describe reaction 8 of CAC:

Answer 40

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.

Question 41

So what are the standard and actual delta G signs of reaction 8?

Answer 41

Standard = +

Question 42

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 ________ .

Answer 42

CO2

Question 43

How many ATP per NADH and FADH2?

Answer 43

2.5, 1.5

Question 44

Indicate how much total ATP from glycolysis, PDC and CAC is received and detail where it comes from

Answer 44

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.

Question 45

Anaerobic bacteria use an incomplete form of the CAC. What is missing and why do they run this?

Answer 45

They do not have a-ketoglutarate dehydrogenase. They use this incomplete CAC for the production of biosynthetic products (AAs, nucleotides, heme, etc.)

Question 46

How did farmers take advantage of anaerobic bacteria ability to use the incomplete cycle?

Answer 46

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.

Question 47

The citric acid cycle is considered to have a ____________ nature

Answer 47

amphibolic

Question 48

Explain the amphibolic nature of the CAC:

Answer 48

This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis.

Question 49

read through amphibolic diagram on slide 33

Answer 49

okay

Question 50

What cycle is found in plants that has CAC intermediates?

Answer 50

Glyoxylate cycle

Question 51

Describe the glyoxylate cycle:

Answer 51

Start is same as CAC to form isocitrate, then you break it up by isocitrate lyase to form glyoxylate and 4C succinate is removed

Question 52

Why is the glyoxylate cycle very important to plants? Explain the importance to things like oil seeds:

Answer 52

Allows for the formation of biosynthetic products by forming succinate, while also not making any CO2.

Question 53

Ultimately, the glyoxysome allows for what to happen?

Answer 53

Convert FA to acetyl coa, then make carbs and proteins

Question 54

Rate controlling enzymes of PDC and CAC:

Answer 54

- PDC

Question 55

Regulation of activity of CAC by: (3)

Answer 55

- Substrate availability

Question 56

Read through slide 37 for what specifically inhibits and activates the 4 enzymes of PDC and CAC regulation.

Answer 56

okay

Question 57

Which reactions of glycolysis, PDC, and CAC produce CO2?

Answer 57

NONE during glycolysis.

Question 58

Carbons have been oxidized to CO2. Completely oxidized without oxygen. Where did the oxygen come from?

Answer 58

The water addition in GAPDH reaction in glycolysis, citrate synthase reaction, and fumarase reaction all contribute the oxygen for oxidation.