Muscle Weakness Module

Question 1

Intermediary Metabolism

Answer 1

All the chemical changes that are involved in the occurance and continuance of life.

Ability to accomplish these changes at constant body temperature requires enzymatic catalysis & thermodynamic coupling of endergonic and exergonic processes.

Question 2

Pathway

Answer 2

The series of steps involved in the breakdown or synthesis of major biological constituents.

Cycle = pathway that regenerates the initial substrate

Catabolic and anabolic pathways linked via ATP.

Question 3

Catabolism

Answer 3

Degradative (complex to simpler)

Oxidative

Exergonic

ATP generating

Often requires NAD⁺ or FAD

Question 4

Anabolism

Answer 4

Sum of the pathways that are involved in synthesis and growth.

Reductive

Energy consuming

ATP utilization

Often requires NADPH

Question 5

Metabolic rate

Answer 5

Expression of enthalpic change.

Gives the normalized total heat production per unit time.

Question 6

Basal metabolic rate

Answer 6

Measured in a resting state (awake laying still)

Energy requirements for:

1. involuntary muscle work
2. maintenance of osmotic gradients
3. maintenance of body temperature
4. turnover and synthesis of cell constiuents

Question 7

Gibb’s free energy

(ΔG)

Answer 7

ΔG = ΔH - TΔS

H= enthalpy S= entropy T= temp in kelvins

The energy change occuring under conditions of constant pressure and temperature.

Additive function and independent of pathway.

Total ΔG of a process can be expressed as the sum of ΔG changes of individual steps.

Question 8

Thermodynamic Coupling

Answer 8

Coupling of an exergonic and endergonic reaction so that net ΔG is negative and reaction can occur.

A common intermediate must exist.

In an enzyme catalyzed reaction, the common intermediate may not be free and may only exist on the enzyme.

Question 9

Standard reduction potentials

(Eº)

Answer 9

The relative affinity of a molecule, atom, or ion for electrons taken under standard conditions where reactants and products are at unit activity (~1 M) then compared to the proton/hydrogen electrode (H+ and ½ H₂)

Often expressed as values corrected to pH 7 (Eº’)

Interpreted as the relative affinity of the system for electrons as compared to that of a proton:

• Negative reduction potential indicates a weaker affinity for electrons than a proton.
• Positive reduction potential indicates a stronger affinity.

Question 10

Cellular reduction potential

Answer 10

Dependent on the ratio of oxidant to reductant agent concentration in a cell.

Question 11

Nernst equation

Answer 11

E = Eº’ + 2.3 RT/nF log [oxidant] / [reductant]

R = gas constant

F = Faraday constant

Relationship of reduction potential to ratio of oxidant to reductant agents in a cell.

Question 12

High energy bonds

Answer 12

Describes a bond which has a large negative standard Gibb’s free energy (ΔGº’) of hydrolysis

Minimum of -7.0 kcal/mol

Indicated with a ~

Question 13

Adenosine triphosphate

(ATP)

Answer 13

• Contains two phosphoanhydride bonds which have a ΔGº’ of hydrolysis of approximately -7.3 kcal/mol each.
• Biological utilization of these high energy bonds require the ATP form.
• One or both bonds may be used in a reaction.
• Phosphoryl group(s) of ATP can be transferred to acceptor molecules to generate activated intermediates for metabolism.
• Can function as coenzyme-cosubstrates.

Question 14

Pyrophosphate

(PP_i)

Answer 14

Use of both phosphoanhydride bonds of ATP is the functional equivalent of using 2 ATP’s.

Reactions coupled to the hydrolysis of the pyrophosphate (PP_i) product to 2 orthophosphates (P_i) by pyrophosphatase which drives reactions forward.

Question 15

Adenylate kinase

Answer 15

Catalyzes the reversible reaction:

AMP + ATP ⇔ 2 ADP

Question 16

Adenine nucleotide cosubstrate metabolic pool

Answer 16

Total concentration of adenine nucleotide pool essentially constant, however, ratio of adenine nucleotides vary with metabolic state of the cell.

Concentrations of ATP, ADP, and AMP in a cell are in rapid equilibrium due to activity of adenylate kinase.

The cosubstrate pool communicates between and significantly influences the different pathways of the cell which utilizes those cosubstrates.

AMP level most sensitive parameter to change in pool and usually initiates the responses to decreased ATP levels.

Question 17

Energy charge

Answer 17

Energy charge = ½ • ( [ADP] + 2[ATP] ) / ( [AMP] + [ADP] + [ATP])

A stoichiometric expression of the mold fraction of high energy phosphate bonds present relative to the maximal high energy bonds possible.

0 = nucleotides are totally in the form of AMP

1 = nucleotides are totally in the form of ATP

Question 18

ΔG for ATP hydrolysis

Answer 18

ΔG_ATP→ADP = ΔGº’_ATP→ADP + RT ln ( [ADP] x [P_i] / [ATP] )

Actual ΔG for ATP hydrolysis in a cell is dependent upon and may be calculated from the concentrations of ATP, ADP, and inorganic phosphate.

May be very different from the ΔGº’.

In a resting cell may be as high as = 15 kcal/mol which is equivalent to an energy charge of approximately 0.9.

“High energy charge”, “large negative ΔG of ATP hydrolysis”, and “resting cell” all indicate that ATP is plentiful.

Question 19

Pathway regulation

Answer 19

• Pathways for synthesis and breakdown of the same constituent are never the same, although individual steps may be reversible and utilized in both pathways.
• Opposing pathways typically occur in seperate cellular compartents and/or different tissues or organs.
• Regulation of pathway enzyme activity by:
  
  • product inhibition
  • allosteric regulation
  • covalent regulation i.e. phosphorylation
• Changes in the rate of synthesis/degradation of an enzyme or its mRNA.

Question 20

Equation for glycolysis

Answer 20

glucose + 2 NAD⁺ + 2 ADP + 2 P_i

→

2 pyruvate + 2 NADH + 2 ATP + 2 H₂O + 2 H⁺

Question 21

General stages of glycosis

Answer 21

1. Priming steps

• Two ATP-linked phosphorylations
• Functions to prevent diffusion of the intermediates of the pathway out of the cell.

2. Oxidation/reduction with the production of NADH
   * Results in the generation of ATP
3. Re-oxidation of the NADH produced.

Question 22

Substrate-level phosphorylation

Answer 22

The synthesis of ATP involving two coupled reactions linked by a common intermediate containing a high-energy bond.

Question 23

Steps of glycolysis

Answer 23

1. Irreversible phosphorylation of glucose at carbon-6 by hexokinase using ATP•Mg²⁺ to produce glucose-6-phosphate.
   
   • Hepatic isozyme is glucokinase.
   • Traps glucose because cell membrane is impermeable to phosphate esters.
   • Commits glucose to intracellular metabolism but NOT glycolysis.
2. Freely reversible aldose-ketose isomerization of glucose-6-phosphate to fructose-6-phosphate by phosphoglucose isomerase.
3. Irreversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate using ATP by phosphofructokinase (PFK1).
   
   • True commitment step and primary regulatory site for glycolysis.
4. Fructose-1,6-bisphosphate split into two triose phosphates: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) by aldolase.
   
   • DHAP freely interconverted to GAP by triose phosphate isomerase.
   • Only GAP enters next stage of glycolysis.
5. Glyceraldehyde-3-phosphate dehydrogenase catalyzes the reversible oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
   
   • NAD+ converted to NADH **
6. Phosphate from the carboxyl group from 1,3-bisphosphoglycerate is reversibly transferred to ADP to produce ATP and 3-phosphoglycerate by 3-phosphoglycerate kinase.
   
   • First substrate-level phosphorylation.
   • Additional mutase found in erythrocytes which transfers carbonyl phosphate of 1,3-bisphosphoglycerate to carbon-2 to produce 2,3-bisphosphoglycerate (2,3-BPG).
7. 3-phosphoglycerate converted to 2-phosphoglycerate by phosphoglycerate mutase.
8. 2-phosphoglycerate converted to phosphoenolpyruvate (PEP) by enolase with loss of H₂O.
9. Phosphate of PEP is transferred to ADP by pyruvate kinase (PK) to produce pyruvate and ATP.
   
   • Irreversible
   • Has a ΔGº’ of -14 kcal/mol
10. NADH produced is recycled back to NAD+
    
    • In aerobic conditions via malate shuttle.
    • In anerobic conditions pyruvate converted to lactate via lactate dehydrogenase.

Question 24

Malate shuttle

Answer 24

Used for the indirect transport of the electrons from glycolysis in the cytoplasm to the respiratory chain in mitochondria.

1. Cytosolic oxidation of NADH coupled with reduction of oxaloacetate to malate.
2. Malate transported across the inner mitochondrial membrane.
3. Malate oxidized back into oxaloacetate as mitochondrial NAD+ is reduced to NADH.

Question 25

Pyruvate kinase (PK) deficiency

Answer 25

* PK-LR gene encodes both RBC and liver isozymes for pyruvate kinase through alternative promoters. * PK deficiency is an autosomal recessive disorder that primarily affects the mature RBC isozyme. * Mutations in coding, splice site, or promoter regions described resulting in an abnormal protein. * Abnormal PK protein has lower affinity for PEP or it's allosteric effectors. * Results in premature distruction (hemolysis) of RBC's leading to hemolytic anemia due to diminished ATP production.

Question 26

Glycolytic isozymes

Answer 26

Transcriptional regulation of glycolytic isozymes used to regulate the flow of glycolysis in order to meet the metabolic needs of rapidly dividing cells (ex. embryonic stem cells or tumor cells). * GLUT-3 transporter and hexokinase (HK-2) up-regulated - both have lower Km for glucose. * Use of pyruvate kinase (PKM2) and lactate dehydrogenase (LDH-A) increases flow of glucose through glycolysis.

Question 27

Fermentation

Answer 27

Glycolysis occurring under anaerobic conditions.

Question 28

Lactate fermentation

Answer 28

Occurs in mammalian tissues specifically skeletal muscle. Pyruvate + NADH → lactate + NAD⁺ Catalyzed by lactate dehydrogenase. Production of NAD⁺ allows glycolysis to continue.

Question 29

Alcoholic fermentation

Answer 29

Occurs in microorganisms such as yeast. **1. Pyruvate → CO₂ + acetaldehyde** Catalyzed by pyruvate decarboxylase. **2. Acetaldehyde + NADH → ethanol + NAD**⁺ Catalyzed by alcohol dehydrogenase.

Question 30

Mitochondrial pyruvate carrier | (MPC)

Answer 30

Transports the pyruvate made via cytoplasmic glycosis through the inner mitochondrial membrane.

Question 31

Stages of cellular respiration

Answer 31

1. Carbohydrates, proteins, and fatty acids are oxidized to two-carbon fragments in the form of acetyl~CoA. 2. The acetyl groups enter the tricarboxylic acid (TCA) cycle which produces CO₂ and the reduced energy carriers NADH and FADH₂. 3. NADH and FADH₂ produced are oxidized in the electron transport chain (ETC) producing H⁺ and electrons. Electrons are transferred to O₂ producing water. Protons used in oxidative phosphorylation to produce ATP.

Question 32

Coenzyme A | (CoA)

Answer 32

* Derived from ATP and pantothenic acid (Vit B5) * Contains a reactive thiol group (-SH) that is covalently linked to the acetyl group via a thioester bond * Acetyl~CoA readily donates acetyl groups to other acceptors

Question 33

Pyruvate dehydrogenase complex | (PDH)

Answer 33

* Member of the α-ketoacid dehydrogenase family * Large multienzyme complex which contains 3 enzymes in multiple copies * Very efficient due to close spatial proximity of complexes **Enzyme subunits** * E1: pyruvate dehydrogenase (aka pyruvate decarboxylase) #30 * E2: dihydrolipoyl transacetylase #60 * Acts as a swinging arm between E1 & CoA preventing substrate from diffusing away ⇒ _substrate channeling_ * E3: dihydrolipoyl dehydrogenase #12 * Contains two stoichiometric coenzymes/cosubstrates * NAD * CoA * Contains three catalytic coenzyme prosthetic groups * thiamine pyrophosphate (TPP) * FAD * lipoic acid * Contains two regulatory enzymes associated with but not part of the complex * PDH kinase * PDH phosphatase

Question 34

Steps for oxidative decarboxylation of pyruvate

Answer 34

1. Pyruvate is decarboxylated and the acetyl group is attatched to thiamine pyrophosphate (TPP) coenzyme of pyruvate decarboxylase (E1). 2. Acetyl group transferred to the lipoic acid covalently bound to dihydrolipoyl transacetylase (E2), 3. The acetyl group, bound as a thioester to the side chain of lipoic acid, is transferred to free CoA. 4. The sulfhydryl form of lipoic acid is oxidized by FAD-dependent dihydrolipoyl dehydrogenase (E3) to regenerate the oxidized lipoic acid. 5. FADH₂ on E3 is reoxidized to FAD as NAD⁺ is reduced to NADH2 + H⁺.

Question 35

Regulation of PDH complex

Answer 35

* Allosteric regulation * Acetyl~CoA and NADH can inhibit complex via allosteric end-product inhibition (feedback) * Covalent modification via phosphorylation and dephosphorylation - main mechanism * E1 inactivated by phosphorylation by PDH kinase * E1 activated by dephosphorylation by PDH phosphatase * Both PDH kinase and PDH phoshatase are in turn allosterically activated by a number of molecules that signal the energy state of the cell * PDH kinase * activated by high-energy signals * ATP * acetyl~CoA * NADH * inhibited by pyruvate * PDH phosphatase * activated by rise in intracellular [Ca⁺⁺] * important in skeletal muscle

Question 36

TCA cycle aka Citric Acid Cycle aka Krebs cycle General

Answer 36

**Acetyl~CoA + 3 NAD⁺ + FAD → 2 CO₂ + 3 NADH + FADH₂** * 8 steps * 4 oxidations that produce NADH or FADH₂ * one substrate-level phosphorylation producing GTP * Except for succinate dehydrogenase which is embedded in the inner mitochondrial membrane all enzymes are located in the matrix

Question 37

Steps of the TCA cycle

Answer 37

1. **Formation of citrate.** * Condensation of acetyl~CoA and oxaloacetate (OAA) catalyzed by *citrate synthase*. * Reaction made **irreversible** by the hydrolysis of the thioester bond of acetyl~CoA. * Facilitated by an enzyme-bound intermediate, citroyl~CoA. * Citrate synthase inhibited by citrate via competitive inhibition. 2. **Isomerization of citrate to isocitrate.** * Citrate isomerized to isocitrate by *aconitase*. * ΔGº' = 6.3 kJ/mol but reaction pushed to the right in vivo because product rapidly consumed in next step. 3. **Oxidation of isocitrate to α-ketoglutarate and CO₂.** * **Irreversible** decarboxylation of isocitrate to α-ketoglutarate by *isocitrate dehydrogenase*. * Loss of CO₂ make the reaction irreversible. * NADH produced. * Isocitrate dehydrogenase inhibited by ATP and NADH. Stimulated by ADP and Ca⁺⁺. 4. **Oxidation of α-ketoglutarate to succinyl~CoA and CO₂.** * Catalyzed by *α-ketoglutarate dehydrogenase complex*. * Same family as PDH dehydrogenase. * Uses same coenzymes/cosubstrates (NAD and CoA) and catalytic coenzymes (TPP, FAD, and lipoic acid) * Inhibited by succinyl~CoA and NADH. * Activated by Ca²⁺. * Not regulated by phosphorylation/dephosphorylation. * NADH produced. * Loss of CO₂ makes reaction **irreversible.** 5. **Conversion of succinyl~CoA to succinate.** * *Succinate thiokinase* catalyzes the hydrolysis of the thioester bond in succinyl~CoA paired to the conversion of GDP to GTP. * GTP interconvertible to ATP by *nucleoside diphosphate kinase*. 6. **Oxidation of succinate to fumarate.** * Catalyzed by *succinate dehydrogenase* with production of FADH₂. * Enzyme located on inner mitochondrial membrane. * ΔGº' = 0 so reaction can go either way but pushed to the right in vivo because fumarate used in next step. 7. **Regeneration of oxaloacetate.** 1. Fumarate converted to malate by *fumarase*. 2. Malate converted to oxaloacetate by *malate dehydrogenase* with production of NADH. * Rxn has a ΔGº' = 29.7 kJ/mol but pulled to the right because OAA used by citrate synthase in step 1.

Question 38

Regulation of the TCA cycle

Answer 38

Regulated almost exclusively at the three irreversible steps. 1. *Citrate synthase* competitively inhibited by citrate. 2. *Isocitrate dehydrogenase* * Inhibited by ATP and NADH * Stimuated by ADP and Ca⁺⁺ 3. *α-ketoglutarate dehydrogenase* 1. Inhibited by succinyl~CoA and NADH 2. Activated by Ca⁺⁺

Question 39

Additional roles of TCA cycle

Answer 39

TCA cycle intermediates can be used for other reactions such as: * amino acid synthesis * fatty acid synthesis * gluconeogenesis

Question 40

TCA cycle intermediate replenishment

Answer 40

Intermediates of the TCA cycle can be provided for by other metabolic pathways, specifically amino acid metabolism.

Question 41

Electron transport chain (ETC) aka Respiratory chain Basics

Answer 41

Inner mitochondrial membrane bound complex. Consists of four seperate protein complexes. Each complex accepts or donates electrons to/from mobile electron carriers (coenzyme Q and cytochrome C). ETC pumps protons from matrix to intermembrane space to form a proton gradient.

Question 42

ETC complexes

Answer 42

* **Complex 1: NADH dehydrogenase** * Flavin mononucleotide (FMN) coenzyme accepts two electrons from NADH to become FMNH₂. * FMNH₂ transfers electrons to CoQ to form CoQH₂. * Enzyme contains iron-sulfur (Fe-S) centers which acts as intermediate electron carriers. * **Complex 2: Succinate dehydrogenase** * Electrons from succinate of TCA cycle transferred via Fe-S centers to FAD to form FADH₂. * **Complex 3: Cytochrome bc₁ reductase** * Electrons from CoQH₂ (from complex 1) are used to reduce cytochrome c which acts as electron carrier. * Contains Fe-S centers which act as intermediates. * Contains heme group which shifts from Fe³⁺ to Fe²⁺ and back as electrons move through. * **Complex 4: Cytochrome oxidase** * Contains two different heme groups * Heme *a* * Heme *a₃* * Contains two Cu ions which act as intermediates * Electrons from cytochrome c transferred to heme *a* via one of the Cu centers, then from heme *a* to heme *a*₃ via other Cu center, and finally to O₂ to form H₂O.

Question 43

Coenzyme Q

Answer 43

Oxidized form ubiquinone reduced to ubiquinol after accepting electron. Accepts electrons from complex I and II of the ETC.

Question 44

Cytochromes

Answer 44

Contain a heme group in which the iron shifts from Fe³⁺ to Fe²⁺ oxidations states and back as electrons move to and from the heme group. All contain the porphyrin ring with differing side chains.

Question 45

Redox potentials | (E'º in volts)

Answer 45

Measures the ease with which an electron can be added or removed. The more positive the value of E'º, the greater the tendancy of the oxidant in the redox pair to accept electrons. Electrons flow from the redox pair with the more negative E'º to one with a less negative/more positive E'º.

Question 46

Relationship of redox potential and free energy change

Answer 46

The redox potential (E'º) is related to the free energy change in the reaction by the Farady constant (F). ΔG'º = -*n*FΔEº *n* = number of electrons transferred ΔEº = difference in reduction potentials of the overall reaction

Question 47

Effective reduction potential | (E)

Answer 47

In the cell, the effective reduction potential (E) depends on the concentration of reactants.

Question 48

Electron flow in ETC

Answer 48

Due to the reduction potentials of each complex the electrons always flow downhill.

Question 49

ETC site-specific inhibitors

Answer 49

Used to block electron flow in ETC. Carriers befor the block become reduced and those after remain oxidized.

Question 50

Oxidative phosphorylation

Answer 50

ATP synthase uses the proton gradient set up by ETC to produce ATP. Dependent on the integrity of the inner mitochondrial membrane. No high-energy intermediate exists (as with substrate-level phosphorylation).

Question 51

Reactive oxygen species | (ROS)

Answer 51

When more electrons enter the ETC than can be immediately passed to O₂ a state of **oxidative stress** exists. Highly reactive superoxide free radicals such as superoxide (•O₂^-) are generated. Mitochondria have systems to eliminate free radicals: 1. Superoxide (•O₂^-) is converted to peroxide (H₂O₂) by *superoxide dismutase.* 2. Peroxide converted to water by *glutathione peroxidase.* NADPH used by *glutathione reductase* to regenerate *glutathione peroxidase.*

Question 52

Respiratory control

Answer 52

The coupling of ATP synthesis and electron transport. Neither process can occur without the other.

Question 53

P:O ratio

Answer 53

The stoichiometry of ATP synthesis relative to the substrate that is oxidized. The amount of ATP formed per ½ O₂ consumed (or per pair of electrons. Approaches 3 for NADH. Closer to 2 for FADH₂ (because succinate oxidation bypasses complex I)

Question 54

Proton pumping by ETC

Answer 54

For each pair of electrons that travel through the ETC and transferred to O₂: Complex 1 pumps out 4 protons Complex 3 pumps out 4 protons Complex 4 pumps out 2 protons **Total of 10 protons per electron pair.** **NADH + 11 H⁺_M + ½ O₂ → NAD⁺ + 10 H⁺_IM + H₂O**

Question 55

Proton motive force | (PMF)

Answer 55

Proton pumping by ETC across the inner mitochondrial membrane sets up a pH gradient (ΔpH) and a transmembrane potential (Δψ) with matrix side negative. Can be calculated as ΔG = 2.3 RT ΔpH + F Δψ In general, transport of a pair of electrons through the ETC generates ~ 53 kcal.

Question 56

Chemiosmotic Hypothesis

Answer 56

The proton motive force drives ATP synthesis as the protons flow passively back through the inner mitochondrial matrix down their concentration gradient through a proton pore in the ATP synthase. The ~ 53 kcal produced per pair of electrons can drive the synthesis of 3 ATPs (using ~ 22 kcal) with the remaining energy used to drive ancillary reactions or dissipated as heat. ADP + P_i + *n*H⁺_IM → ATP + H₂O + nH⁺_M Value of *n* depends on the structure of the ATP synthase and varies between species (~3.3 - 5 protons per ATP)

Question 57

Mitochrondrial ATP synthase | (Complex V)

Answer 57

**F₁F_o ATPase** O stands for oligomycin which blocks the proton pore F-type ATPase which functions as a reversible ATP-driven proton pump * F₁ portion contains the ATPase domain. * Contains 9 subunits α₃β₃γδε * α and β subunits for the knoblike catalytic section * γ subunit forms a shaft that connects with F_o * F_o complex consists of a, b, and c subunits * 8 c-subunits form the c-ring in vertebrates * Rotation of C-ring induces conformational changes in the β subunits of F₁ that drive ATP synthesis * Requires 8 protons moving across the membrane with each rotation and produces 3 ATP's per turn

Question 58

Yield of ATP production

Answer 58

* Takes **11 H⁺ to generate 3 ATP's** * 8 H⁺ to give a complete c-ring rotation * Additional 3 H⁺ to bring 3 phosphates into the matrix via the mitochondrial phosphate transporter * From 1 NADH: 10 H⁺ pumped so (10/11) x 3 ATPs produced = **2.75 ATPs per NADH** * From 1 FADH₂ (Succinate): 6 H⁺ pumped so (6/11) x 3 ATPs produced = **1.64 ATPs per FADH₂ ** * Glycolysis generates 2 net ATPs and 2 NADH * PDH generates 1 NADH/pyruvate → 2 NADH/glucose * TCA generates: * 3 NADH/pyruvate → 6 NADH/glucose * 1 FADH₂/pyruvate → 2 FADH₂/glucose * 1 ATP/pyruvate → 2 ATP/glucose * OxPhos: * 10 NADH → 27.5 ATP * 2 FADH₂ → 3.3 ATP Going all the way through oxidative phosphorylation generates: * ~ 31 ATP by OxPhos * 2 ATP for glycolysis * 2 ATP for TCA **TOTAL OF ~35 ATP/GLUCOSE**

Question 59

Respiratory inhibitors

Answer 59

CN^- or CO blocks electron transport and subsequently ATP synthesis

Question 60

Oligomycin

Answer 60

Phosphorylation inhibitor Blocks ATP synthesis and thus electron transport

Question 61

Uncouplers

Answer 61

**dinitrophenol (DNP)** **uncoupling protein 1 (UCP1 aka thermogenin)** * Causes collapse of the proton motive force releasing energy in the form of heat rather than ATP. * Electrons free to move through the ETC. * Increases oxygen consumption.

Question 62

Regulation of oxidative phosphorylation

Answer 62

ETC and ATP synthesis coupled so increasing or decreasing one does the same to the other.

Question 63

Ancillary reactions

Answer 63

All the transport processes associated with oxidative phosphorylation which utilize the proton motive force or transmembrane potential. Mostly used to transport molecules across the impermeable inner mitochondrial membrane. * Agents that disrupt the PMF will reduce transport. * Transport of these substances will conversely reduce the PMF available for ATP synthesis. * Electroneutral transport systems driven by concentration gradients alone. * Electrogenic transport systems utilize concentration gradients as well as the transmembrane potential. * positively charged molecules enter matrix more easily * Adenine nucleotide translocase * Phosphate translocase * Transporters for other charged metabolites such as pyruvate, malate, citrate, etc. * Ca⁺⁺ and asparate also transported in a PMF-dependent fashion

Question 64

Adenine nucleotide translocase

Answer 64

Antiporter that exchanges ADP^3- from the intermembrane space for ATP^4- from the matrix. Net transport of 1 negative charge out of the matrix (electrogenic) stimulated by the matrix-negative transmembrane potential. Inhibited by atractyloside.

Question 65

Phosphate translocase

Answer 65

Symporter that moves one phosphate (H₂PO₄^- ) and one proton (H⁺) into the matrix. Driven by the proton gradient established by ETC. Electronically neutral.

Question 66

ATP synthasome

Answer 66

Complex containing ATP synthase, adenine nucleotide translocase, and phosphate translocase can be isolated by gentle disruption of the inner mitochondrial membrane suggesting that these three proteins are spatially integrated.

Question 67

Nicotinamide nucleotide transhydrogenase

Answer 67

A transmembrane protein embedded in the inner mitochondrial membrane. Uses the proton gradient to drive: **NADH + NADP⁺ + H⁺_IM → NAD⁺ + NADPH + H⁺_M** NADH is used by ETC so its levels are related to the potential for ROS generation. Production of NADPH by transhydrogenase will increase as more electrons travel down ETC. NADPH indirectly used in ROS neutralization.

Question 68

Malate-Aspartate Shuttle

Answer 68

Found in most tissues. 1. Electrons from NADH transferred to cytosolic oxaloacetate using *cytosolic* *malate dehydrogenase* to form malate. 2. Malate enters the matrix via malate-α-ketoglutarate transporter. 3. Inside the matrix, malate converted back to oxaloacetate by *mitochondrial* *malate dehydrogenase* with concomitant reduction of NAD⁺ to NADH. 4. Matrix oxaloacetate converted to aspartate and sent back to the cytosol where it is metabolized to oxaloacetate, completing cycle.

Question 69

Glycerol-3-phosphate shuttle

Answer 69

Used in skeletal muscle and brain. 1. NADH generated in glycolysis used by *cytosolic glycerol-3-phosphate dehydrogenase* to convert dihydroxyacetone phosphate to glycerol-3-phosphate. 2. Glycerol-3-phosphate enters the matrix where it is converted back to DHAP by *mitochondrial* *glycerol-3-phosphate dehydrogenase* producing FADH₂ which enters ETC at CoQ. Since FADH₂ produced instead of NADH the yield of ATP is diminished.

Question 70

Mitochondrial genes

Answer 70

Mitochondria contain their own genome which contains 37 genes. 13 encode subunits of respiratory chain proteins.

Question 71

Mitochondrial diseases

Answer 71

Maternally inherited. Defects in oxidative phosphorylation most commonly associated with mutations in mitochondrial genes. Presumably due to generation of reactive oxygen species. Affects tissues with high requirement for ATP such as brain, liver, and skeletal/cardiac muscle.

Question 72

Leber's hereditary optic neuropathy | (LHON)

Answer 72

* Caused by a mutation in one of the subunits of Complex I of ETC which renders it unable to transfer electrons from NADH to CoQ. * Patients with LHON can only use electron transfer via succinate (complex II) thus produce less ATP. * Leads to bilateral loss of vision in early adulthood.

Question 73

Mitochondria and apoptosis

Answer 73

* Triggered by: * External signals acting via a receptor * Oxidative stress * Heat shock * Viral infection * Exposure to stimulus induces formation of large pores in the outer mitochondrial membrane called permeability transition complex * Allows release of cytochrome c into the cytosol * Cytochrome c in associated with apoptosis protease activating factor 1 (Apaf-1) activate a family of cytosolic proteases (the caspases) that degrade proteins and lead to cell death.

Question 74

Glycogen Structure & Function

Answer 74

* Major short-term storage form of carbohydrates in animals. * For times of metabolic need. * Branched chain homopolysaccharide of α-D-glucose * Primary bond is α-1,4-glycosidic linkages. * Every 8-10 glucose residues there is a branch attached via a α-1,6-glycosidic linkage * Stored in the cytoplasm as large hydrated granules. * Each granule contains as many as 55,000 glucose units. * Found in the cytoplasm of liver and skeletal muscle cells primarily.

Question 75

Sources of blood glucose

Answer 75

1. Diet 2. Degradation of glycogen 3. Gluconeogenesis

Question 76

Liver Glycogen

Answer 76

* Functions to maintain the blood glucose concentration particularly during the early stage of a fast. * Glucose rapidly released from liver glycogen. * Glucose able to enter systemic circulation due to presence of _glucose-6-phosphatase_ in the liver. * Hepatic glycogen stores ~24 hour supply of glucose. * Liver also able to synthesize glucose via _gluconeogenesis_.

Question 77

Skeletal Muscle Glycogen

Answer 77

* Serves as a fuel reserve for synthesis of ATP that will power muscle contraction. * Muscle glycogen is not available to other tissues because muscle lacks glucose-6-phosphatase.

Question 78

Methods of Activating Sugars

Answer 78

1. Phosphorylation * Glycolysis utilizes glucose-6-phosphate. 2. Create a nucleotide sugar * Glycogenesis utilizes UDP-glucose. Alternate activation methods allows for both pathways to occur at the same time.

Question 79

Glycogenesis Step 1: Chain Synthesis

Answer 79

* Occurs in the cytoplasm and can be divided into two stages: 1. Chain synthesis 2. Chain branching I. Chain Synthesis **A. Synthesis of UDP-glucose.** 1. _Glucose_ is phosphorylated to _glucose-6-phosphate_. - By *glucokinase* in hepatic tissue. - By *hexokinase* in peripheral tissue. 2. _Glucose-6-phosphate_ converted to _glucose-1-phosphate_ by *phosphoglucomutase*. 3. _Glucose-1-phosphate_ reacts with _UTP_ to form _UDP-glucose_ and _PP_i_ which is catalyzed by *glucose 1-phosphate uridylyltransferase* (aka *UDP-Glc pyrophosphorylase*) - Hydrolysis of _PP_i_ → _2 P_i_ by *pyrophosphatase* makes the reaction energetically favorable and irreversible. **B. Requirement of primer to initiate glycogen synthesis.** ⇒Glycogen synthase cannot initiate glycogen synthesis de novo and can only add glucose to an existing chain, therefore, glycogenesis requires a primer. 1. _Glycogen_ _fragment_ can serve as a primer for *glycogen synthase* which attaches glucosyl residues to existing chain using _UDP-glucose_. 2. The protein *_glycogenin_* (homodimer) can prime glycogen synthesis and attach glucose residues through auto-glucosylation ⇒ serves as both substrate and enzyme in its role as primer. a. The *glycosyltransferase* activity of _glycogenin_ transfers the first molecules of glucose from _UDP-glucose_ to a specific _tyrosine side-chain_ (tyr-194) on itself. b. After at least 4 (about 7) glucose residues have been added, *glycogen synthase* takes over. - Glycogenin remains within the glycogen granule. **C. Elongation of glycogen chains.** 1. *Glycogen synthase* transfers glucose from _UDP-glucose_ to the _non-reducing end of the growing chain_ via α-1,4-linkages between the -OH group on C-1 of the activated sugar and the C-4 of the accepting sugar. ⇒ *Glycogen synthase* is the rate-limited and regulated enzyme of glycogenesis. ⇒ There are liver & muscle isozymes. ⇒ _UDP_ released when α-1,4-glycosidic bond is formed can be converted to _UTP_ by *nucleoside diphosphate kinase* + ATP.

Question 80

Glycogenesis Step 2: Chain Branching

Answer 80

* Occurs in the cytoplasm and can be divided into two stages: 1. Chain synthesis 2. Chain branching II. Chain branching A. Catalyzed by "branching enzyme" called *glucosyl 4:6 transferase.* 1. *Glucosyl 4:6 transferase* cleaves an _α-1,4-glycosidic bond_ from the non-reducing end of the glycogen chain producing a 6-8 glucosyl residues fragment. 2. Enzyme then transfers the fragment to another residue on the linear chain via an _α-1,6-glycosidic bond_. 3. Resulting new non-reducing end and old non-reducing ends can be further elongated by *glycogen synthase*. 4. After further elongation, new chains of 6-8 residues can be transferred to make additional branches.

Question 81

Functions of branching in Glycogen

Answer 81

1. Increases solubility of glycogen molecule. * Stored as hydrated granules. 2. Increases number of non-reducing ends to which new glucosyl residues can be added to or removed from glycogen. * Facilitates fast breakdown of glycogen into glucose when energy needed.

Question 82

Glycogenolysis Overview

Answer 82

* Occurs primarily in the cytoplasm of liver and skeletal muscle cells. * Involves 2 stages: 1. Shortening of chains 2. Removal of branches

Question 83

Glycogenolysis Step 1: Shortening of Chains

Answer 83

1. *Glycogen phosphorylase* uses **Pi** to cleave the α-1,4-glycosidic bonds between glucose residues at the non-reducing ends of the glycogen chains releasing _glucose-1-phosphate_. ⇒Enzyme is a homodimeric exoglucosidase ⇒Requires *pyridoxal phosphate (PLP)* coenzyme (derivative of Vit B6) ⇒Liver and muscle isozymes * *Glycogen phosphorylase* stops attacking α-1,4-glycosidic bonds _four glucosyl residues from an α-1,6-branch point_. * Resulting structure is called a **limit dextrin.** 2. **​**_Glucose-1-phosphate_ is converted to _glucose-6-phosphate_ by *phosphoglucomutase*. 3. Next step in glycogenolysis depends on the tissue: * In liver: _glucose-6-phosphate_ is hydrolyzed to _free glucose and P_i_ by *glucose-6-phosphatase*. ⇒Free glucose able to leave the liver and enter the blood stream. * In peripheral tissues: glucose-6-phosphate will be oxidized in the glycolytic pathway to produce energy.

Question 84

Glycogenolysis Step 2: Removal of branches

Answer 84

Catalyzed by a debranching enzyme which is a bifunctional protein with two catalytic activities. 1. *4-α-D-glucantransferase* activity transfers the _outer three glucosyl residues_ of the _limit dextrin_ to a _non-reducing end_ by breaking and formation of α-1,4 bonds leaving one glucosyl residue in α-1,6 linkage. ⇒ 4,4 transferase activity 2. _α-1,6 linkage_ is then cleaved hydroytically by the *amylo-α-1,6 glucosidase* activity of debranching enzyme releasing _free glucose_ (non-phosphorylated). 3. Co-operative and repetitive action of phosphorylase and debranching enzymes results in complete hydrolysis of glycogen to yield glucose-1-phosphate and free glucose in a 12:1 ratio. ⇒Free glucose is quickly phosphorylated in muscle for intracellular use. ⇒Phosphorylated glucose is already trapped inside muscle cells.

Question 85

Regulation of Glycogen Metabolism

Answer 85

_Key regulatory enzymes of glycogen metabolism:_ Glycogen synthase (synthesis) & Glycogen phosphorylase (degradation) Each is controlled by: 1. _Hormone-induced covalent modification_ through phosphorylation or dephosphorylation of ser residues. * Way of responding to the needs of the body as a whole. 2. _Allosteric effectors_ * Way of responding to the need of a particular tissue at a particular time.

Question 86

Glycogen Phosphorylase Covalent Regulation (Control of glycogen breakdown)

Answer 86

**I. Regulation by covalent modification:** Glycogen phosphorylase exists in two forms: _A_ or active form: phosphorylated _B_ or inactive form: dephosphorylated A and B forms are interconverted by: *Phosphorylase kinase* Produces active phosphorylated A-form. & *Phosphoprotein phosphatase-1* Produces inactive dephosphorylated B-form. \*The A form of glycogen phosphorylase is more active because phosphorylation causes a conformational change in the enzyme which shifts the equilibrium of the enzyme between its T-state (taut and inactive) and R-state (relaxed and active) towards the active R-state. The B-form is inactive because the taut state is favored. *Phosphorylase kinase* (regulatory enzyme) is itself regulated by phosphorylation/dephosphorylation. A and B forms are interconverted by: *Protein Kinase A (PKA)* Produces active phosphorylated A-form. & *Phosphoprotein phosphatase-1* Produces inactive dephosphorylated B-form.

Question 87

Hormonal Control of Glycogen Breakdown

Answer 87

Hormonal signals that activate PKA include Glucagon and Epinephrine. Phosphorylase kinase (regulatory enzyme) & Glycogen phosphorylase (regulated enzyme of glycogenolysis) are phosphorylated in response to hormonal signals that are transduced via cAMP which activates protein kinase A. PKA also results in the **inhibition** of *phosphoprotein phosphatase-1* by phosphorylating and activating *protein phosphatase inhibitor* (A-form). This enzyme is used to maintain the level of phosphorylation until hormone signal changes. Active protein kinase A has a short half-life because seperation of regulatory and catalytic subunits revealed PEST sequences which marks protein for degradation.

Question 88

Glucagon

Answer 88

Peptide hormone released from the alpha-cells of the pancreas when blood glucose is low. Glucagon binds to plasma membrane receptors on liver cells but not muscle. Stimulates glycogen degradation via PKA-mediated activation of phosphorylase kinase. Activated during periods of fasting thus making glucose available to tissues.

Question 89

Epinephrine

Answer 89

Hormoned released by the adrenal medulla in response to physiological stress. Epinephrine binds to β-adrenergic receptors on the plasma membrane of both liver and muscle cells. Stimulates glycogen degradation via cAMP and PKA mediated phosphorylase kinase activation.

Question 90

Glycogen Synthase Covalent Regulation (Control of glycogen synthesis)

Answer 90

*Glycogen synthase* exists in two forms: A or active form: dephosphorylated B or inactive form: phosphorylated Several kinases phosphorylate glycogen synthase A to the inactive B form including: * cAMP dependent PKA* * Phosphorylase kinase* * Glycogen synthase kinase-3* * AMP-dependent kinase* B-form converted back into the active A-form by *Phosphoprotein phosphatase-1.* PKA also results in the inhibition of phosphoprotein phosphatase-1 by phosphorylating and activating *protein phosphatase inhibitor* (A-form).

Question 91

Reciprocal Regulation of Glycogen Synthesis & Degradation

Answer 91

cAMP regulates glycogen metabolism through the simultaneous activation of glycogenolysis and inhibition of glycogenesis. Also displays amplification: 1 hormone activates many adenyl cyclase, makes many cAMP, each activates many PKA, each phosphorylates many phosphorylase kinase, each phosphorylates many phosphorylase ect.

Question 92

Insulin

Answer 92

Released by pancreas in response to high blood glucose levels. Has the opposite affect of glucagon/epinephrine. Activates glycogen synthesis and inhibits degradation in liver and muscle. 1. Promotes inhibition of several protein kinases and activation of phosphoprotein phosphatase. * Causes subsequent dephosphorylation of glycogen synthase (activating) and dephosphorylation of phosphorylase kinase and phosphorylase (inactivating). 2. Promotes conersion of cAMP to 5' AMP by activating a phosphodiesterase. * Causes subsequent decrease in active PKA.

Question 93

Allosteric Regulation of Glycogen Synthesis

Answer 93

* Allosteric effectors are superimposed onto covalent regulation in order to meet the needs of the tissue. * Enzymes of glycogen metabolism including glycogen phosphorylase kinase, glycogen phosphorylase, and glycogen synthase are regulated in an allosteric effectors acting in a _non-covalent manner_. * ​Postive allosteric effectors bind to a regulatory site on the R (active) form of the enzyme stabilizing it and pulling the equilibrium towards the R-form. * Negative allosteric effectors bind to the T (inactive) form and stabilizes that pulling equilibrium towards T-form. * Effectors include: * Ca²⁺ and AMP which are signs of low energy * Glucose and glucose-6-phosphate which are signs of high energy

Question 94

Calcium Mediated Regulation of Glycogen Metabolism

Answer 94

Released in times of energy need.​ Ca²⁺ binds to the calmodulin-like δ-subunits of dephosphorylated (B-form) *phosphorylase kinase* causing conformational change which activates the catalytic γ-subunits in the absence of phosphorylation ⇒ stabilizes the R-state Ca²⁺ also required for maximal activation of phosphorylase kinase a. *Phosphorylase kinase* subsequently phosphorylates and inhibits *glycogen synthase*. End result of a rise in [Ca²⁺]_in​ is increased degradation and decreased synthesis of glycogen. * **Contracting muscle** * Ca²⁺ released in in response to nerve impulses. * [Ca²⁺]_in​ activates sarcoplasmic glycogenolysis by activating phosphorylase kinase. * **Liver cells** * Epinephrine binds to α-adrenergic receptors, activating phospholipase-C, and generating IP₃ and DAG from PIP₂. * IP₃ causes release of Ca²⁺​ from the SER. * [Ca²⁺]_in​ activates glycogenolysis by activating phosphorylase kinase⇒ activates glycogen degradation. * DAG activates PKC which phosphorylates and inactivates glycogen synthase ⇒ inhibites glycogen synthesis. Side note: Ca²⁺​ also activates mitochondrial events: * Activates PDH phosphatase thereby activating PDH and pyruvate degradation. * Allosteric effector of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase thereby activating TCA cycle.

Question 95

AMP Mediated Regulation of Glycogen Metabolism

Answer 95

* AMP to ATP ratio reflects the energy state in muscle cells. * Increase in AMP signals low energy and need for glycogen degradation. * AMP functions as an allosteric activator of muscle phosphorylase b. * Directly activates the dephosphorylated form of myophosphorylase b.

Question 96

Glucose Mediated Regulation of Glycogen Metabolism

Answer 96

* When glucose is plentiful, _heptatic_ glycogenolysis is decreased by glucose itself acting as an allosteric inhibitor of hepatic phosphorylase a. * The glucose-6-phosphate fromed from glucose is an allosteric activator of glycogen synthase b in _both liver and muscle_, thus increasing glycogenesis. * For this reason, glycogen synthase b is sometimes designated "D" because it is dependent on glucose-6-phosphate for activity while synthase a is designed "I" for independent.

Question 97

Hypoxic Response

Answer 97

* Under normal oxygen conditions: * _Prolyl hydroxylase domain (PHD) enzyme_ hydroxylates two prolines in the oxygen-dependent degradation domain (ODDD) of HIF-1α * Rxn requires Fe²⁺ and Vit C * Hydroxylated HIF-1α is a candidate for von-Hippel-Lindau (VDL) protein which ubiquitonates it * Ubiquinated HIF-1α degrated by proteasomes * _Factor Inhibiting HIF-1_ (FIH) hydroxylates C-domain of HIF-1α * Hydroxylation prevents binding of HIF-1α transcription coactivator p300 * Under hypoxic conditions: * PHD and FIH enzymes inactive * HIF-1α has NSL which allows entry into nucleus * Dimerizes with HIF-1β * HIF-1 response elements (HREs) * Complex binds to HIF-1 response elements (HREs) * Upregulates vascular endothelial growth factor (VEGF) and other factors to stimulate angiogenesis * Upregulates erythropoietin (EPO) to stimulate erythropoiesis * Transcriptionally re-programs glycolysis to increase glucose uptake and increase the output of ATP. * Up-regulation of lactate dehydrogenase to allow for the regeneration of NAD⁺

Question 98

GSD Type Ia

Answer 98

**Von Gierke Disease** * **Glucose-6-phosphatase deficiency** * Liver and kidney * Severe fasting hypoglycemia hallmark * Major Findings * Hepato/renomegaly * Fasting hypoglycemia * Lactic acidemia * Hyperuricemia * Hyperlipidemia

Question 99

GSD Type Ib

Answer 99

**Von Gierke Disease** * **ER glucose-6-phosphate transporter deficiency** * Liver and kidney * Recurrent infections as a result of neutropenia * Major Findings * Hepato/renomegaly * Fasting hypoglycemia * Lactic acidemia * Hyperuricemia * Hyperlipidemia

Question 100

GSD Type II

Answer 100

**Pompe Disease** aka Generalized Glycogenosis * **Lysosomal acid α-glucosidase deficiency** * Infantile, juvenile, and adult-onset forms * Affects all organs but skeletal/cardiac most * Cardiomegaly/myopathy in infantile forms * Muscle weakness in later forms * Enzyme replacement therapy has reduced mortality

Question 101

GSD Type III

Answer 101

**Cori Disease** aka Limit Dextrinosis * **Debranching enzyme deficiency** (both actions) * IIIa : affects liver and muscle * IIIb: affects liver only * Milder hepatomegaly * Muscle weakness * Accumulated glycogen has abnormal structure with shorter chains * May cause liver fibrosis or cirrhosis

Question 102

GSD Type IV

Answer 102

**Andersen Disease** aka Amylopectinosis * **Branching enzyme deficiency** in liver * Progressive hepatomegaly * Accumulated glycogen has abnormal structure with longer chains and no branches * Progressive liver cirrhosis in infantile form can be lethal

Question 103

GSD Type V

Answer 103

**McArdle Disease** * **Muscle glycogen phosphorylase deficiency** * Infantile and Adult forms * Exercise intolerance and muscle cramps * Symptoms usually first appear in adolescence * _Failure of blood lactate to rise_ after anaerobic exercise

Question 104

GSD Type VI

Answer 104

**Hers disease** * **Liver glycogen phosphorylase deficiency** * Hepatomegaly * Benign in general * Mild fasting hypoglycemia

Question 105

GSD Type VII

Answer 105

**Tarui Disease** * **Muscle PFK-1 deficiency** * Affects muscle and RBC's * More severe exercise intolerance and muscle cramps * RBC's show some percentage of normal activity