citric acid cycle

Question 1

what is cellular respiration?

Answer 1

the main way energy is produced. cells consume O2 and produce CO2, providing lots of energy ATP and captures energy stored in lipids and amino acids. used by animas, plants, and many microorganisms

Question 2

three stages of respiration and what they generate (energy)

Answer 2

acetyl CoA production: generates some ATP, NADH, and FADH2
acetyl CoA oxidation: generates more NADH, FADH2, and one GTP
electron transfer and oxidative phosphorylation: generates tons of ATP

Question 3

where do glycolysis, CAC, and oxidative phosphorylation take place in the cell?

Answer 3

glycolysis = cytoplasm
CAC = mitochondrial matrix
oxidative phosphorylation = inner mitochondrial membrane

Question 4

respiration step 1

Answer 4

conversion of pyruvate to acetyl CoA
net reaction: oxidative decarboxylation of pyruvate. first carbons of glucose to be fully oxidized (produces CO2)
enzyme: pyruvate dehydrogenase complex. uses 5 coenzymes (TPP, lipyllysine, FAD, NAD+, and CoA-SH) which ones are prosthetic and which are co-substrates?
reactants: pyruvate
products: CO2, acetyl CoA, NADH

Question 5

details of pyruvate dehydrogenase complex PDC structure

Answer 5

PDC is a multi enzyme complex with three enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoyl dehydrogenase (E3).
Advantages of multi enzyme complex: allows channeling of substrates, minimizing side reactions, and allows regulation of entire complex from activity of one subunit

Question 6

draw the overall reaction of PDC, remember each subunit and cofactors

Answer 6

slide 17

Question 7

draw the mech for TPP. what is the purpose of the N? what is the part of TPP that is important for chemistry?

Answer 7

chemistry occurs at the thiazolium ring. the N accepts electron pair and allows decarboxylation. slide 12

Question 8

what other reaction have we seen before that uses TPP? how is it different in respiration?

Answer 8

Ethanol fermentation uses TPP to convert pyruvate to acetaldehyde. the difference is just the absence of lipoyllysine at the last step.

Question 9

what is important to know about lipoyllysine structure?

Answer 9

it has a very long linker that allows intermediates to move long distances

Question 10

what is the function of Coenzyme A? what is the functional part of the molecule?

Answer 10

to accept and carry acetyl groups. the reactive thiol group is what functions in chemical reactions

Question 11

list the 8 steps in the CAC

Answer 11

1. C-C bond formation to make citrate
2. Isomerization via dehydration/rehedration
3. oxidative decarboxylation to give NADH
4. 2nd oxidation decarboxylation to give NADH
5. substrate level phosphorylation to give GTP
6. dehydrogenation to give reduced FADH2
7. hydration
8. dehydrogenation to give NADH

Question 12

CAC step 1

Answer 12

C-C bond formation (only C-C bond formation step) by condensation of acetyl-CoA and oxaloacetate
reactants: acetyl CoA, oxaloacetate
products: citrate
enzyme: citrate synthase
uses acid/base catalysis
rate limiting step
activity depends on [oxaloacetate]
spontaneous/irreversible 
mech slide 24

Question 13

how does citrate synthase avoid unnecessary hydrolysis of thirster in acetyl-coA?

Answer 13

citrate synthase has an induced fit. upon oxaloacetate binding, it changes from the open conformation (which does not allow acetyl-CoA binding) to the close conformation (which does allow acetyl-CoA binding). so acetyl coA is not hydrolyzed unless oxaloacetate is present

Question 14

CAC step 2

Answer 14

isomerization by dehydration/rehydration
reactants: citrate
products: isocitrate
enzyme: aconitase
citrate is a poor substrate for oxidation, isocitrate is good for oxidation (tertiary vs secondary alcohol)
addition of H2O is stereospecific to cis-aconitate
nonspontateous: [P] kept low

Question 15

what part of aconitase catalyzes the reaction?

Answer 15

the iron-sulfur center. the sulfurs come from Cys. the center is sensitive to oxidative stress

Question 16

how is aconitase stereospecific?

Answer 16

the active site has complementary binding points that make citrate orient the correct way for only R isocitrate to be produced. slide 28

Question 17

CAC step 3

Answer 17

oxidative decarboxylation
reactants: isocitrate. NADP+
products: a-ketoglutarate, NADPH, CO2
enzyme: isocitrate dehydrogenase
loses C as CO2 (most oxidized C, no more reductions possible) and generates NADPH
spontaneous/irreversable: regulated by product inhibition

Question 18

CAC step 4

Answer 18

final oxidative decarboxylation
reactants: a-ketoglutarate, CoA-SH, NAD+
products: succinyl-CoA, CO2, NADH
enzyme: a-ketoglutarate dehydrogenase complex
Net full oxidation of all Cs in glucose (after 2 cycles) although carbons lost actually came from oxaloacetate
enzyme complex similar to PDC, only differ in substrate sizes
spontaneous/irreversible: regulated by P inhibition
slide 32-33, 17 for mech

Question 19

CAC step 5

Answer 19

generation of GTP through thioester
reactants: succinyl-CoA, GDP, Pi
products: succinate, GTP, CoA-SH
enzyme: succinyl-CoA synthetase
energy of thioester allows for incorporation of inorganic phosphate. goes through phospho-enzyme (His) intermediate
non spontaneous: [P] kept low
mech slide 37

Question 20

CAC step 6

Answer 20

oxidation of an alkane to alkene
reactants: succinate, FAD
products: fumarate, FADH2
enzyme: succinate dehydrogenase
bound to mitochondrial inner membrane as part of ETC complex. FAD is unusually covalently bound so FADH2 must be oxidized to restore (cofactor, not catalytic)
near equilibrium, reversible: [P] low
mech slide 38

Question 21

CAC step 7

Answer 21

hydration across a double bond
reactants: fumarate
products: L-malate
enzyme: fumarase
stereospecific, addition of water is always trans to form L-malate. 
slightly reversible: [P] low
mech slide 40

Question 22

CAC step 8

Answer 22

oxidation of alcohol to ketone
reactants: L-malate, NAD+
products: oxaloacetate, NADH
enzyme: malate dehydrogenase
final step regenerates oxaloacetate
highly unfavorable/reversible: [oxaloacetate] kept very low
mech slide 42

Question 23

net result of CAC (1 turn)

Answer 23

acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O –> 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
net oxidation of two carbons to CO2, equivalent to two C from acetyl-CoA but actually form oxaloacetate.
energy captured by electron transfer to NADH and FADH2 (which generate lots of ATP through ETC) and generates one GTP (=ATP)

Question 24

what does it mean the CAC intermediates are amphibolic? (same thing as anaplerotic??)

Answer 24

they can be removed and put back into the cycle at any intermediate. cycle just needs to have equal input of carbons as the number of carbons lost.
the 4 C intermediates can be formed by carboxylation of 3 C precursors. slide 48 for examples

Question 25

how is the CAC regulated?

Answer 25

regulation occurs at highly thermodynamically favorable/irreversible steps (PDH, citrate synthase, IDH, and KDH)
general mechanism for all enzymes: activated by substrate availability (NAD+ and AMP), inhibited by product accumulation (NADH and ATP)

Question 26

how is pyruvate dehydrogenase complex regulated?

Answer 26

by reversible phosphorylation of E1 where phosphorylation makes it inactive. PDH kinase and PDH phosphorylase are part of the complex. kinase is activated by ATP
high ATP = active kinase phosphorylates PDH = less acetyl-CoA
Low ATP = kinase is less active, phosphorylase removes phosphate from PDH = more acetyl-CoA

Question 27

how is citrate synthase regulated?

Answer 27

inhibited by succinyl-CoA which is an indicator of flow at a-ketoglutarate branch point. if succinyl-CoA is high, cycle is inhibited early on at citrate synthase

Question 28

how does isocitrate dehydrogenase regulate steps as far back as PFK in glycolysis?

Answer 28

regulating IDH will control citrate levels. aconitase is reversible so inhibition of IDH leads to accumulation of isocitrate and reverses aconitase, accumulating citrate. the citrate leaves the mitochondria and inhibits PFK-1 in glycolysis

Question 29

how is the glyoxylate cycle different from the CAC?

Answer 29

it has 2 C-C bond forming steps and results in no GTP and only 1 NADH
bypasses decarboxylation with two enzymes: isocitrate lyase and malate synthase.

Question 30

why is the glyoxylate cycle used?

Answer 30

in plants and some microorganisms (glyoxysome). it is not energy producing, but it feeds into the CAC. It has a net production of 2 acetyl-CoA –> oxaloacetate which allows for net conversion of acetyl CoA to glucose.