Terminal Oxidation Pathways

Question 1

Describe transport of fatty acids into mitochondria by carnitine

Answer 1

• Outer membrane of mitochondria= porins (protein pores) so acyl-CoA can enter
• Inner membrane very impermeable to acyl-CoA
• Acyl group transferred from coenzyme A onto carnitine= acyl- carnitine, catalysed by carnitine palmitoyl transferase 1 on outer membrane, inhibited by malonyl-CoA
• Acyl-carnitine imported through inner membrane in exchange for free carnitine
• Back to coenzyme A after transport so acyl-CoA

Question 2

Describe fatty acid oxidation

Answer 2

-Once in mitochondria, acyl-CoA oxidised by beta-oxidation pathway
-Enzymes of beta oxidation pathway within mitochondrial matrix, catalyse the oxidation of long-chain fatty acids
-Fatty acid shortened by 2 carbons with acetyl-CoA split off
-Repetitive rounds= shortened
-If odd number of carbons= produces a propanyl-CoA
=Double bond introduced
=Reduction of electron-transferring flavoprotein which has FAD prosthetic group
=Reduces ubiquinone coenzyme
=Double bond hydrated to form hydroxy-acid attached to CoA, oxidised by NAD to form oxo-acid
=Acetyl-CoA split off

Question 3

Compare fatty acid synthesis and degradation

Answer 3

• Synthesis= cytoplasm, reductive, NADPH coenzymes, multienzyme complex, intermediates esterified to enzyme complex
• Degradation= mitochondria, oxidative, NAD+ and ubiquinone coenzymes, separate enzymes, CoA-esters intermediates

Question 4

Describe oxidation of propionate

Answer 4

-Propionyl Co-A from odd number fatty acids or metabolism of isoleucine, valine, methionine and a cholesterol side chain (bile acid synthesis)
=Carboxylated then isomerized (involves B12 as coenzyme
=Succinyl CoA, an intermediate in tricarboxylic acid cycle

Question 5

What is the pyruvate dehydrogenase complex?

Answer 5

-Pyruvate formed by glycolytic pathway in cytoplasm
-Pyruvate dehydrogenase in mitochondrial matrix= 30 copies of one type of subunit, 60 and 12 of others
=Thiamine pyrophosphate (vitamin B1) prosthetic group= first step catalyst
=Transacetylase has lipoic acid
=Dehydrogenase that reoxides enzyme contains flavin adenine dinucleotide derived from B2

Question 6

Describe pyruvate oxidation

Answer 6

-Pyruvate crosses inner mitochondrial membrane via pyruvate transporter
=Oxidative decarboxylation of pyruvate
=oxidised to acetyl group which is esterified onto coenzyme A, loss of CO2, NAD reduced to NADH

Question 7

What causes thiamine deficiency?

Answer 7

• Dietary deficiency
• Beri-Beri
• Common in alcoholics as inhibits uptake of vitamin B1 and its processing into coenzyme thiamine pyrophosphate
• Symptoms= tremor, paralysis

Question 8

What does alcoholic metabolism cause?

Answer 8

-Thiamine deficiency
-Hypoglycaemic crises because it inhibits gluconeogenesis
=Treated by infusion of glucose
-Vitamin B1 deficiency

Question 9

How is pyruvate dehydrogenase activity regulated?

Answer 9

• Phosphorylation/ dephosphorylation cycle
• Pyruvate dehydrogenase kinase phosphorylates subunit type 1
• Activated by HADH, ATP and acetyl-CoA (products of pyruvate oxidation that inhibit enzyme)
• Pyruvate dehydrogenase phosphatase activates enzyme by dephosphorylation activated by calcium and insulin

Question 10

What is the TCA cycle?

Answer 10

• Tricarboxylic acid cycle in mitochondrial matrix
• Glucose to pyruvate to acetyl-CoA as does fatty acids, ketogenic amino acids
• Enters cycle by reaction with oxaloacetate to form citrate (irreversible)
• Three dehydrogenases reduce NAD: isocitrate (CO2 lost), oxoglutarate (CO2 lost), malate
• Succinate dehydrogenase reduces ubiquinone (coenzyme, not soluble in water)
• Conversion of succinyl-CoA to succinate linked to formation of GTP

Question 11

What are the gluconeogenic amino acids converted into in the TCA cycle?

Answer 11

=glutamic acid, histidine, proline and arginine converted to oxoglutarate
=valine, isoleucine, threonine and methionine converted into succinyl CoA
=phenylalanine and tyrosine to fumarate (inner mitochondrial membrane)

Question 12

Describe the stoichiometry of the TCA cycle

Answer 12

-One turn= acetyl-CoA completely burnt up to 2 CO2, 3 NADH, reduced ubiquinone and a GTP
=10 or 11 ATP (10.6)

Question 13

Describe ketone bodies

Answer 13

• Acetoacetate and 3-hydroxy butyrate produced in liver, produced during starvation
• Acetyl CoA formed from beta oxidation builds up, diverted to ketone body formation in mitochondria
• HMG-CoA intermediate broken down to acetoacetate, reduced to 3-hydroxy butyrate
• Circulate in plasma, oxidised as fuel
• Acetoacetate can be spontaneously decarboxylated (not stable) to produce acetone
• High concentration= acid-base disturbance (strong acids so metabolic acidaemia)

Question 14

What happens to coenzymes in the TCA cycle?

Answer 14

• Reduced
• Re-oxidised by electron transport chain
• Electrons reduce oxygen to
• Oxygen is terminal electron acceptor
• Electron transport release energy, conserved for ATP synthesis by ATP synthase

Question 15

Describe the mitochondrial ultra-structure

Answer 15

• Outer membrane permeability barrier for proteins, porins
• Infoldings of inner membrane increase surface area= cristae
• Inner membrane more impermeable to small molecules, carriers, electron transport chain are integral membrane proteins in inner
• Oxidative enzymes in mitochondrial matrix

Question 16

What is an electron transport chain?

Answer 16

• Series of carriers, pass electrons to one another
• Reduced by accepting electrons from donor
• Movement is from carriers of lower affinity for electrons to higher affinity
• Expressed as redox potential

Question 17

Describe the structure of the prosthetic groups of the electron transport chain

Answer 17

• Attached to proteins covalently or by strong non-covalent forces
• Flavoproteins= flavin prosthetic, derived from vitamin B2 or riboflavin, 2 forms= flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), accepts 2 H to become reduced (hydrogen carrier)
• Iron-sulphur clusters= +side chains of cysteines in the protein, accepts electrons, single electron carrier, iron from ferric to ferrous form

Question 18

Describe ubiquinone

Answer 18

• Coenzyme free in the mitochondrial inner membrane
• Synthesised not derived on same pathway as cholesterol
• Very hydrophobic, poly-isoprenoid side chain, dissolved in inner membrane, hydrogen-carrier

Question 19

What are cytochromes?

Answer 19

• Iron atom coordinated by series of four 5-membered rings called haem
• Different types depending on side-chains on rings, may be covalently or non-covalently attached to proteins
• Cytochrome c= smallest and simplest, haem covalently attached to cysteine side chains
• B-type haem present in haemoglobin, reversibly binds to oxygen, always in ferrous state, no redox function

Question 20

Examples of electron transport chain inhibitors

Answer 20

• Rotenone= plant derived poison (insecticide)- Complex 1
• Atovaquone= complex 2, component of antimalarial drug Malarone
• Cyanide and CO= complex 4, block electron transport to oxygen

Question 21

What do the complexes do in the electron transport chain?

Answer 21

• Complex 1 oxidises reduced NAD and reduces ubiquinone
• Complex 2 is succinate dehydrogenase, reduces ubiquinone
• Complex 3 reoxidizes ubiquinone, reducing cytochrome c on outside of inner membrane
• Complex 4 reoxidizes cytochrome c, reduces oxygen to water
• Complex 5 makes ATP (ATP synthase), does not have an redox prosthetic groups

Question 22

Describe proton translocation by mitochondrial electron transport chain

Answer 22

• Proton circuit through mitochondrial membrane (chemiosmotic theory)
• Electron transport chain translocate protons out of the mitochondrial matrix, setting up electrochemical gradient of protons (proton-motive force)
• Small difference in pH (1.5 units, higher inside mitochondria), membrane potential (150 millivolts) because protons positively charged
• Source of energy for ATP synthase as protons return through ATP synthase (inhibitor= oligomycin)

Question 23

What are uncouplers?

Answer 23

• Protons short-circuit, pumped out but do not make ATP when they come back in, energy released as heat (damage to mitochondria)
• Chemicals that induce this effect, lipid-soluble weak acids which bind protons on the outside, diffuse through the membrane and release them inside

Question 24

What is the P/O ratio?

Answer 24

Defined as the number of molecules of ATP produced per atom of oxygen reduced
The molecules of ATP per 2 electrons moving along the chain
-For chemiosmotic theory= number of protons from reducing one oxygen divided by the number of protons you need to make an ATP
-For NADH oxidation, 10 protons translocated by chain, ATP synthase makes 3 ATPs from translocation of 8 protons back into mitochondria so= 2.5 (take into account proton required for phosphate import)
-For succinate oxidation, 6 protons translocated from reoxidation of ubiquinone= 1.5

Question 25

Describe the control of oxidation by the demand for ATP (respiratory control)

Answer 25

- Contracting muscle- myosin-ATPase hydrolyses ATP to ADP and phosphate - Change in ratio of ATP to ADP stimulates ATP synthase= increased proton translocation= ATP synthase is working faster= more protons are translocated back =lowers the electrochemical gradient of protons= lowers protonmotive force= stimulates electron transport chain= rate of NADH oxidation increases= increases ratio of oxidised to reduced NAD= activates TCA cycle