Lecture 20 - Midterm 3

Question 1

What are the three stages of cellular respiration?

Answer 1

Stage 1: generation of acetyl CoA and a pair of electrons

Stage 2: oxidation of the two carbon atoms from acetyl CoA in the Citric Acid Cycle to form two CO2 molecules and four pairs of electrons.

Stage 3: comprises electron transport and oxidative phosphorylation

During this final stage the reduced electron carriers that were generated from Stages 1 and 2 become re-oxidized, thus providing energy for the synthesis of ATP

Question 2

Where does the citric acid cycle occur?

Answer 2

– mitochondrial matrix

Question 3

T or F, acetyl-CoA production occurs in the mitochondria, then enters the CAC

Answer 3

True

Question 4

Where does ETC and oxidative phosphorylation occur?

Answer 4

– inner membrane of mitochondria

Question 5

What is the 6 carbon molecule that is generate from Oxaloacetate and Acetyl CoA in CAC

Answer 5

– Citrate

– there’s a total of 8 reactions in the CAC, including four dehydrogenation reactions, which generate eight reducing equivalents (3 NADH/H + and FADH2)

Question 6

T or F, when it comes to the oxidation of Acetyl CoA to 2 CO2, cleaving a C-C bond occurs more easily when Carbonyl groups are present to stabilize carbanion transition states

Answer 6

True

Question 7

what is the intermediate compound that is formed before citrate is created?

Answer 7

– citroyl CoA

Question 8

What is the citrate synthase - reaction mechanism?

Answer 8

– upon frmation of Citroyl CoA, the CoA group is cleved by hydrolysis, and citrate is then released

– 2-part reaction: condensation followed by hydrolysis –> catalyzed by citrate synthase. Key point is that the energy stored in the thioester bond is used to synthesize a larger molecule from smaller precursors; allos reaction to proceed to right

– good example of how enzymes not only promote the correct reaction, but also prevent wasteful side reactions (e.g. hydrolysis of acetyl CoA without it’s energy)

Question 9

T or F, proton transfers on acetyl CoA activate nucleophilic enol for attack on carbonyl group of OAA

Answer 9

True; once nucelophilic enol attacks OAA, this produces intermediate, citroyl CoA;

– spontaneous hydrolysis of citroyl CoA causes cleaving of Co - A - SH group resulting in Citrate

Question 10

What are the key properties of citrate synthase?

Answer 10

– ordered kinetics and extreme conformational changes to the enzyme upon reactant binding –> leads to adoption of “closed” conformation (this is required 4 acetyl CoA binding)

– oxaloacetate binds first and this causes the binding site to be transferred to the core of the protein. Further conformational shift creates the acetyl CoA binding site

–

Question 11

T or F, induced fit is key to preventing wasteful hydrolysis of acetyl CoA

Answer 11

true; catalytic residues required for hydrolysis are not available until after citroyl CoA has already been made

Question 12

Why does the hydroxyl group need to be moved for decarboxylation?

Answer 12

– needs to be moved to allow a carbonyl to form

– citrate is a tertiary alcohol bound to hydroxyl group and 3 other carbons issue bc tertiary alcohols cannot be oxidized w/o breaking C-C bond

– this is bc carbon atom bound to OH group is already bount to 3 other carbons and thus cannot form carbon-oxygen bond

– therefore to combat OH being in wrong place, it will have 2 be shifted around to allow carbonyl (carbon-oxygen) to form

Question 13

What needs to happen in order for hydroxyl group to be shifted around?

Answer 13

– enzyme aconitase will catalyze a dehydration/hydration reaction

– hydrosyl group along w/ another hydrogen molecule leave citrate in form of water (dehydration rxn) forming cis-Aconitate and remains enzyme bound

– then water is combine w/ cis-Aconitate (hydration rxn) to form D-Isocitrate

Question 14

Where does the CCA occur? and what does the oxidation of isocitrate to alpha ketoglutarate generate?

Answer 14

– matrix of mitochondria

– NADH, release of oxidized CO2

Question 15

What enzyme catalyzes the oxidation of isocitrate to alpha ketoglutarate and what is the reaction mechanism?

Answer 15

– isocitrate dehydrogenase

– 2 hydrogens from isocitrate are going to be removed and donated to NAD+, it is then temporarily converted to oxalosuccinate

– there is a decarboxylation that happens and then carbon dioxide is released before it is converted to alpha ketoglutarate

– energy that Is released is going to be used for the next reaction in order to drive the cycle in a forward direction

Question 16

What oxidizes alpha ketoglutarate ?

Answer 16

– alpha ketoglutarate dehydrogenase

– mechanistically, this reaction, is very similar to what was studied for the pyruvate dehydrogenase complex

– high energy electron carrier being generated; since it’s a significant amount of energy the carrier is NADH

– the most energy that is released in the cycle

Question 17

How does the conversion of succinyl CoA to succinate generates ATP (or GTP)

Answer 17

– this time the high-energy thioester bond is used to drive phosphorylation of ADP to form ATP (or GDP to GTP), depending on the isoform of the enzyme

– only point in cycle where ATP or GTP is created; doesn’t matter which one is made

– anytime you see synthetase ATP is going to be generate

Question 18

What catalyzes the conversion of succinyl CoA to succinate generates GTP (or ATP)?

Answer 18

– catalyzed by succinyl CoA synthetase ( removes phosphate off succinyl phosphate which is an intermediate and then adds phosphate to ADP or GDP)

– only TCA cycle step that produces compound w/ high phosphoryl-transfer potential

– cells can use GTP as an energy source as it is readily converted to ATP

Question 19

What is the reaction mechanism of the conversion of succinyl CoA to succinate?

Answer 19

– Step 1: orthophosphate reacts with succinyl CoA to form succinyl phosphate

– Step 2: active site histidine then captures phosphoryl group and succinate released

– Step 3: Phosphohistidine then swings away and interacts with GDP bound to active site, generating GTP and resetting enzyme

– succinyl CoA synthetase –> good example of energy transformation

Question 20

T or F, succinate serves as foundation for regeneration of oxaloacetate

Answer 20

True; methylene group is converted to the carbonyl group in oxaloacetate, and along the way more high-energy electrons are captured (by NADH and FADHs)

Question 21

What are the 3 reactions that that are included in the conversion of succinate to oxaloacetate?

Answer 21

3 reactions: oxidation then hydration then oxidation again

1st rxn: converts succinate to fumarate and generates FADH2; relative to NADH, FADH2 is a minor contributor during oxidative phosphorylation, FAD is hydrogen acceptor in this rxn b/c the free-energy change is not sufficient to reduce NAD+ to NADH

2nd rxn: converts fumarate to malate by hydration; NADH is a higher electron carrier than FADH –> meaning it has higher energy and contributes more to ATP synthesis

3rd rxn: converts malate to oxaloacetate and resets the TCA cycle ; only rxn in the cycle with a large positive free-energy change; rxn is driven to right by utilization of rxn products: NADH is consumed during ox-phos & oxaloacetate is consumed by citrate synthase at beginning of TCA cycle

Oxaloacetate will be consumed and used up and allows reaction to move forward

Question 22

What is the overall summary of the Rxns of the TCA Cycle

Answer 22

1: 2 C atoms enter the cycle during condensation of acetyl CoA with oxaloacetate. 2 C atoms leave in the form CO2
2: 4 pairs of H atoms leave the cycle in 4 oxidation reactions to generate 3 NADH and 1 FADH2. Remember that 1 NADH is also produced in generating acetyl CoA from pyruvate
3: 1 ATP/GTP is produced
4: 2 molecules of water are consumed

– NADH carries 2 electrons at a time, a total of 8 electrons leaving CAC

Question 23

T or F, the overall operation of the reaction of CAC proceeds in a unidirectional way due to the 3 reactions with highly negative delta G

Answer 23

True

Question 24

What are the net products of the CAC?

Answer 24

• • 2CO2
• • 3 NADH
• • FADH2
• • GTP
• • CoA
• • 3 H+

Question 25

What are anaplerotic reactions?

Answer 25

– many intermediates of the CAC are used in biosynthetic reactions

– side reactions happen in order to make sure oxaloacetate isn’t used up

– in order to keep the CAC running and make sure that the intermediates aren’t used up, apalerotic reactions are used to “fill-up” the intermediates –> or make intermediates

Question 26

T or F, transmination reactions can make Oxaloacetate from Aspartate (and other amino acids)

Answer 26

True

Question 27

What is the committed step in the Citric Acid cycle?

Answer 27

Acetyl-CoA; once it’s made it allows cycle to occur and it’s irreversible

Question 28

Why can’t you trace the carbons at succinate?

Answer 28

– because of the symmetry, one can’t tell it looks all the same

– the carbons come from acetyl CoA

Question 29

What is the critical branch point in TCA?

Answer 29

– the pyruvate dehydrogenase complex, it’s irreversible and rate-limiting critical branch-point for all of metabolism

Question 30

Why is CAC regulation more complex?

Answer 30

– because it is a source of metabolic intermediates as well as energy

– entry point and irreversible reactions are both highly regulated, as in Glycolysis

*note: Acetyl CoA is used in other reactions through lipid biogenesis/metabolism

Question 31

Explain the regulation of pyruvate dehydrogenase (PDH)

Answer 31

– Regulation by feedback inhibition.

– the products of the pyruvate dehydrogenase reaction, acetyl CoA and NADH, inhibit pyruvate oxidation if allowed to accumulate

– NADH and Acetyl CoA act as competitive inhibitors bec if you have a lot of them there’s no point in making more

Question 32

What is the primary mechanism for control of PDH in mammals?

Answer 32

– covalent modification of E1

– phosphatase activated and turns PDH ON in response to insulin or muscle contractions or epinephrine signal (removes phosphate from PDH activating it)

– a kinase and a phosphatase inactivate and activate the first component E1 of the PDH complex by phosphorylation and dephosphorylation, respectively, three specific serine residues

– kinase is activated and turns OFF PDH when products of the reaction accumulate (takes phosphate from ATP and adds phosphate to PDH, deactivating it)

Question 33

Describe the regulation of the CAC.

Answer 33

– Allosteric regulation and substrate availability at exergonic reactions

– Citrate synthase: allosterically by NADH and Succinyl-CoA (comp.). NAD+ availability (substrate).

– Isocitrate dehydrogenase is allosterically activated by ADP and inhibited by NADH and ATP. NAD+ substrate availability is also key

– alpha keto glutarate dehydrogenase is also negatively regulated by its reaction products (succinyl CoA and NADH). Competitive inhibitors