Bioenergetics

Question 1

Reference reading for bioenergetics

Answer 1

REFERENCE READING: Medical Biochemistry. An Illustrated Review. Panini, SR. Thieme, c2013, Pages 127 -148.

• Disorders of the Krebs cycle – see supplied PDF (reading material-highlighted sections)
• Genetic alterations in Krebs cycle and its impact on cancer pathogenesis – see supplied PDF (reading material-highlighted sections)

Question 2

Explain the importance and regulate of pyruvate dehydrogenase complex (PDC)

Answer 2

Phosphorylated form of PDC is inactive • Phosphorylation occurs in coenzyme Thiamine pyrophosphate (TPP) of E1 complex • Dephosphorylated version of PDC is active

In a phosphatase deficiency  PDC is always in phosphorylated form (inactive)  Glucose  Lactate rather than Acetyl CoA  Results in constant lactic acidosis  (i.e., high blood levels of lactic acid)  Central nervous system is effected the most  Alanine intake should be restricted

(p. 128‑129; Table 10.1, Fig. 10.2)

Question 3

Explain PDC in tissues

Answer 3

Correlation box

Question 4

Explain Pyruvate dehydrogenase deficiency (neonatal lactic acidosis)

Answer 4

correlation box (p. 129)

Question 5

What is the significance of Arsenite and lipoic acid?

Answer 5

correlation box (p. 129)

Question 6

Describe Beriberi and Wernicke-Korsakoff Syndrome [Students should comprehend the importance of thiamine in the function of PDC]

Answer 6

correlation box (p. 129)

Question 7

Compare and contrast the TCA cycle reaction steps and identify where high-energy equivalent substances are formed

Answer 7

 Steps 1 and 2 : Condensation and isomerization to generate isocitrate  Step 3 and 4 : Irreversible oxidation and decarboxylation NADH, CO2 , and Succinyl CoA  Step -5: Formation of Succinate and GTP  Step 6 -8: Produce FADH 2 , NADH, and regenerate Oxaloacetate  Steps 1,3, and 4 : Regulated steps

 When cellular ATP levels are low, the activity of TCA cycle is increased  High levels of cellular ATP triggers the inhibition mechanism of TCA cycle  mitochondrial ETC inhibition

(Fig. 10.3, p. 130-131)

Question 8

Describe Differential effects of citrate on rate-limiting enzyme

Answer 8

correlation box (p. 130)

Question 9

What are the ATP:ADP and NADH/NAD+ ratios?

Answer 9

correlation box (p. 132)

Question 10

How does rat poison inhibit the TCA cycle?

Answer 10

correlation box (p. 133)

Question 11

Describe succinyl CoA in heme synthesis

Answer 11

(p. 133)

Question 12

Compare and contrast the anaplerotic reactions and anabolic functions of TCA cycle. Describe how the TCA cycle switches to make intermediates in fasting and fed state.

Answer 12

• Anaplerotic “fill up” reactions provide intermediates for replenish TCA Cycle • Two major anaplerotic reactions 1. Degradation of amino acids 1. Carboxylation of Pyruvate

(p. 133‑135; Fig. 10.4; Fig. 10.5 A-B)

Question 13

Describe the effects of Pyruvate carboxylase deficiency

Answer 13

(p. 134)

Question 14

Discuss disorders of TCA cycle

Answer 14

Mitochondrial depletion syndrome associated with  Profound hypotonia  Progressive dystonia  Muscular atrophy  Severe sensory neural hearing impairment

(see reading reference)

Question 15

Describe Oxoglutaric acid aciduria

Answer 15

2-Oxoglutaric aciduria (a-ketoglutaric acid) A rare disorder with global developmental delay and severe neurological problems in infants  Metabolic acidosis  Severe microcephaly  Mental retardation

reading reference

Question 16

What happens in Fumarase deficiency?

Answer 16

Characterized by severe neurological impairment. Fatal outcome within the first 2 yrs. of life  Encephalomyopathy  Dystonia  Increased urinary excretion of fumarate, succinate, aketoglutarate, and citrate  Autosomal recessive disorder  Mutation in fumarase gene contains Q319E

reference reading

Question 17

Succinyl CoA synthetase deficiency

Answer 17

Succinyl-CoA synthetase (SCS) deficiency  Associated with mutations two out of three subunits making up the enzyme, SUCLA2 and SUCLG1  These genes encode the β-subunit of the ADP-forming SCS and α-subunit of SCS  Oncometabolites of TCA cycle are Citrate and 2-hydroxyl glutarate

reference reading

Question 18

What are the Oncometabolites of the TCA cycle?

Answer 18

Oncometabolites are small biomolecules (or enantiomers) of normal metabolism. Excessive accumulation of them causes metabolic dysregulation [91]. Consequently, cells with high oncometabolite concentrations undergo progression to cancer [91]. Oncometabolites of the TCA cycle are accumulated due to mutations of genes involved in metabolism.

The oncometabolites can be divided in two categories - (I) native oncometabolites that could be accumulated due to mutations (loss of function) of genes encoding the enzymes, and (ii) promiscuous oncometabolites that could be accumulated due to gain-of-function mutations in metabolic enzymes.

reference reading

Question 19

Compare and contrast free radicals as oxidizing agents, signaling molecules, and cellular response to invading microorganisms

Answer 19

(Fig. 12.18, p. 194)

Question 20

Define the respiratory chain and discuss the mechanisms of OxPhos and Components of respiratory chain

Answer 20

A successful OxPhos must accomplish the following key goals : 1. To transfer electrons from NADH and FADH2  O2

2. To establish a proton gradient across the inner mitochondrial membrane and in intermembrane space
3. To synthesize ATP
   (p. 138; Fig. 11.2; p. 140-143) AND (Table 11.1)

Question 21

Cytochrome-c and apoptosis

Answer 21

correlation box (p. 137)

Question 22

What are the Respiratory chain components encoded by mtDNA?

Answer 22

correlation box (p. 144)

Question 23

What is Rotenone?

Answer 23

correlation box (p. 144)

Question 24

Cyanide poisoning

Answer 24

correlation box (p. 144)

Question 25

Describe Cyanide versus carbon monoxide action

Answer 25

correlation box (p. 144)

Question 26

Describe Aspirin overdose

Answer 26

correlation box (p. 145)

Question 27

What is Electron transfer?

Answer 27

1)-Transfer of Electrons  The electron flows from the molecules with lower E0 ’ to that with highest E0 ’  The difference in Δ E0 ’ is associated with ΔG0’  Δ E0 ’ and ΔG0’ are inversely related  ΔG0’ = -nƑ Δ E0 ’

Inhibition of electron transport • When transfer of electrons inhibited A decrease in the pumping protons A decrease in the proton (H+) gradient Inhibition of ATP synthesis

reference reading

Question 28

Proton gradient and proton motive force (Δ pmf)

Answer 28

 Electron transfer through the respiratory chain lead to the pumping of H+ from matrix to the innermitochondrial space  Two factors constitutes a proton-motive force (pmf) to drive ATP synthesis by Complex V 1) pH gradient (ΔpH) 2) Membrane potential (ΔΨ)

reference reading

Question 29

Chemiosmosis theory- Compare and contrast coupling and uncoupling of proton gradient and is consequences.

Answer 29

Uncoupling [disruption of protein gradient]  In OxPhos  Electron transfer couples to proton gradient  P ~ADP  ATP

If the proton gradient is disrupted  P ~ ADP uncouples from the electron transfer  Protons (H+) reenter the mitochondrial matrix from the intermembrane space  TCA cycle and electron transfer to O2 are accelerated  ATP synthase is inhibited (No ATP Synthesis)  Heat generation

(p. 142; Fig. 11.6).

Question 30

Compare and contrast the mitochondrial electron transport chain (ETC) toxins

Answer 30

(p. 142; Fig. 11.6)

Question 31

Synthesis of ATP

Answer 31

3)- Synthesis of ATP • Catalyzed by a large membrane-bound protein • ATP synthase (Complex V) • Harnesses the energy contained in pmf, obtains necessary power (7.3 kcal/mol) to form ADP + Pi  ATP Inhibitors: Oligomycin disrupts proton transport through the channel

reference reading

Question 32

Regulation of OxPhos

Answer 32

 Generates most of the ATP  It is sensitive to O2 [ATP/ADP] ratio

(p. 143; Fig. 11.7)

Question 33

Thermogenesis in brown adipose tissue

Answer 33

(p. 144)

Question 34

Other electron transport systems

Answer 34

correlation box (p. 145)

Question 35

Hypoxia and ATP preservation

Answer 35

correlation box (p. 145)

Question 36

Nucleotide analogue treatment and fatigue

Answer 36

correlation box (p. 145)

Question 37

Antioxidants and removal of free radicals

Answer 37

Antioxidants • Superoxide dismutase (SOD) O-. 2 + 2H+ O2 + H2O2 • Catalase 2H2O2 O2 +2H2O • Glutathione peroxidase • Vit-E • Vit-C

Superoxide dismutase 1. Cu/Zn SOD (SOD1) cytosolic 2. Mn/Zn SOD (SOD2) mitochondrial

reference reading

Question 38

Free radicals and antioxidants

Answer 38

Correlation box (p. 10)

Question 39

Ubiquinone radical

Answer 39

Correlation box (p. 145)

Question 40

Explain the mitochondrial transport system. Compare and contrast tissue specific shuttle systems

Answer 40

(p. 146-148).

Question 41

Malate-Aspartate shuttle system

Answer 41

Malate-aspartate shuttle Operates in the heart, liver, and kidneys Generates NADH in mito-matrix NADH enters to ETC at Complex-I

(p. 147; Fig. 11.8 A)

Question 42

Glycerophosphate shuttle system

Answer 42

Glycerophosphate-shuttle  Operates in skeletal muscle and brain  Generates FADH2 in the inner mitomembrane  FADH2 joins to ETC at CoQ

(p. 147; Fig. 11.8 B)

Question 43

Antiporters for phosphate / OH¯ and phosphate / malate exchange

Answer 43

(p. 148; Fig. 11.9 A, B)

Question 44

Antiporter for ADP / ATP exchange

Answer 44

(p. 148; Fig. 11.9 C)

Question 45

Antiporter for pyruvate / OH¯

Answer 45

(p. 148; Fig. 11.9 D)

Question 46

Sources of NADH and FADH2

Answer 46

Correlation Box (p.145)

Question 47

Other electron transport systems

Answer 47

Correlation Box (p. 145)

Question 48

Discuss mitochondrial medicine: An overview

Answer 48

reference reading

Question 49

First mitochondrial disorder; Luft’s

Answer 49

disease reference reading

Question 50

Causes of mitochondrial disease

Answer 50

• Primary cause : – Defect in nuclear DNA (nDNA) encoding the mitochondrial proteins – Defect in mitochondrial DNA (mDNA) • Secondary causes: – Ischemia – Reperfusion – Cardiovascular diseases – Renal failure – Drugs and aging – Alcohol – Smoking. – Others

reference reading

Question 51

Defect in nuclear DNA (nDNA)

Answer 51

reference reading

Question 52

Defect in mitochondrial DNA (mDNA)

Answer 52

reference reading

Question 53

Manifestations of mitochondrial disease

Answer 53

Manifestations of mitochondrial diseases

• Clinical features – Nervous system: seizures,ataxia,dementia, deafness, blindness – Eyes : ptosis, external ophtalmolplegia,retinis pigmentosa with visual loss – Skeletal muscle : Muscle weakness, fatigue, myopathy, exercise intolerance, loss of coordination and balance – Heart: cardiomyopathy – Others: gastrointestinal, liver failure, kidneys, pancreatic disease, diabetes, etc.
• Metabolic Features – Low energy production – Increased free radical production – Lactic acidosis

reference reading

Question 54

TCA cycle and cancer

Answer 54

Studies demonstrated that abnormalities of the TCA cycle are associated with different types of cancers [25e27] (Fig. 1). For example, it was noted that phosphoenolpyruvate carboxykinase, a regulator of TCA cycle flux, plays a crucial role in promoting cancer cell growth and proliferation, particularly in colorectal cancer cells [27]. Phosphoenolpyruvate carboxykinase increases glucose and glutamine uptake in cancer cells and favours anabolic metabolism [27]. This metabolic shift is important for highly proliferating cells, like cancer cells, which continuously require a supply of precursors for the synthesis of lipids, proteins and nucleic acids [28]. Several studies also demonstrated that dysfunction of mitochondrial metabolic pathways such as the TCA cycle and oxidative phosphorylation are associated with the conversion of non-neoplastic mammary epithelial cells to non-metastatic breast carcinoma and then to the metastatic cancer cells [29e31].

Question 55

Describe the role of excess Citrate in cancer.

Answer 55

Excess citrate reduces the activity of the mitochondrial isoform of pyruvate dehydrogenase and results in a shift of the cell’s metabolism towards glycolysis [94]. Increased accumulation of pyruvate leads cells to convert pyruvate to lactate and regenerate NAD þ which is the key factor for glycolysis [94] (Fig. 1). Thus citrate accumulation further favours the non-oxidative breakdown of glucose in the cells and promotes cancer growth.

Increased accumulation of citrate activates the enzyme acetyl CoA carboxylase (ACC), which in turn increases the production of acetyl CoA and malonyl CoA [97,98]. This increased acetyl CoA and malonyl CoA is then directed towards increased lipid/sterol synthesis [97,98]. The transformed lipid metabolism observed in cancer cell shifts the metabolic fate of lipid synthesis of the cell membrane and changes the redox potential of cells. These alterations promote cellular processes like cell growth, proliferation, cell survival signalling and differentiation which in turn contributed in the tumorigenesis [99].

Question 56

Describe the role of 2-Hydroxy glutarate (2HG) in cancer

Answer 56

. 2-Hydroxy glutarate (2HG) Mutations of IDH1 and IDH2 (cytosolic and mitochondrial isoforms of IDH) lead to the accumulation of 2HG and acts as a pathogenic metabolite (oncometabolite) in many cancers [42,52,106e110]. A study also reported that this mutation was involved in the conversion of alpha-ketuglutarates to R (-)-2-hydroxyglutarates (2HGs) [41]. Excessive accumulation of these 2HGs acts as an oncometabolite and led to the malignant progression of gliomas