(L4) Mitochondrial Bioenergetics Flashcards
What is CK-MB and what is its clinical significance?
L4 LO1
Panini pg. 114, blue box
Phosphorylates creatinine to form phosphocreatinine, an energy storage molecule that replenishes ATP.
CK-MB is found in muslce and brain tissues and can be detected in the blood at elevated levels following an MI.
What are the high energy substances produced from the TCA cycle and what is their ATP equivalent?
L4 S6 LO2
- NADH (2.5 ATP)
- FADH2 (1.5 ATP)
- GTP (1 ATP)
What common molecule do fats, carbohydrates, and proteins enter the TCA?
What is this molecule made from?
What does it do?
What is its deta G?
L4 S6 LO2
Acetyl CoA
It is synthesized from pantothenic acid, atp and cystine
-It forms a high energy thioester linkage with acyl groups such as acetate or fatty acids
- The linkage in acetyl COA has a delta G of -7.5 kcal/mol
- ATP is -7.3 kcal/mol
How does pyruvate enter into the mitochondria?
L4 S8
- pyruvate must enter the outer membrane of the mitochondria via a porin then through the inner membrane via a mitocondrial pyruvate carrier (MPC)
- PDC will transform it into A-COA
What is the pyruvate dehydrogenase complex and what are its cofactors?
L4 S9
Trimer:
- E1
- E2
- E3
Cofactors:
- thiamine pyrophosphate (TPP) (E1)
- lipoic acid (E2)
- CoA (E2)
- FAD (E3)
- NAD+ (E3)
**** The first three are prosthetic groups that are permanently bound to PDC while the other two are cosubstates and are only associated with the PDC temporarily
all are derived from vit b except for lipoic acid
What is the function of citrate synthase?
L4 S10 LO2
Takes acetyl CoA (2), oxaloacetate (4), and water to make citrate (6).
What does aconitase do?
L4 S10 LO2
Converts citrate (6) to isocitrate (6).
What does isocitrate dehydrogenase do?
What special feature does this reaction have?
L4 S10 LO2
Dehydrogenates isocitrate (6) to form intermediate of oxalosuccinate which is then is decarboxylated to form α-ketoglutarate (5).
Dehydrogenation converts NAD+ to NADH and H+.
CO2 is produced from decarboxylation.
***rate limiting step***
What does α-ketoglutarate dehydrogenase complex do?
What special feature does this reaction have?
L4 S10 LO2
Converts α-ketoglutarate (5) to succinyl CoA (4).
Forms high energy thioester bond.
Decarboxylation produces CO2.
Hydrogen from thiol of CoA converts NAD+ to NADH.
What does succinyl CoA synthase do?
What special feature does this reaction have?
L4 S10 LO2
Converts succinyl CoA (4) to succinate (4).
Produces GTP through phosphate level phosphorylation using high energy thioester bond with CoA.
What does succinate dehydrogenase do?
What special features does this reaction have?
L4 S10 LO2
Converts succinate (4) to fumarate (4) via dehydrogenation.
Dehydrogenation converts FAD to FADH2.
Enzyme is located in inner mitochondrial membrane.
FADH2 is not released from the enzyme as the electrons are passed to Co-Q (complex 2) in the electron transport chain.
What does fumarase do?
L4 S10 LO2
Converts fumarate (4) to L-malate (4) via hydration of double bond.
What does malate dehydrogenase do?
L4 S10 LO2
Dehydrogenates malate (4) to form oxaloacetate (4) and H+.
Dehydrogenation converts NAD+ to NADH.
What enzymes of the citric acid cycle have regulatory mechanisms?
L4 S9;11 LO2
- pyruvate dehydrogenase
- citrate synthase
- isocitrate dehydrogenase
- α-ketogultarate dehydrogenase
All are inhibited by high [ATP]
What is Coenzyme A?
L4 LO3
Panini pg. 123, orange box
Derived from:
- pantothenic acid (vitamin B5)
- ATP
- cysteine
Used to activate acyl groups (C=O) using high energy thioester linkage
What enzynes and molecules regulate pyruvate dehydrogenase activity?
L4 S9 LO4
Phosphorylated PDC is inactive
PDC directly inactivated by:
-Acetyl CoA, NADH
Pyruvate dehydrogenase kinase (PDK), inactivating:
- stimulated by:
- Acetyl CoA, NADH, ATP
- inhibited by:
- CoA, NAD+, ADP
Pyruvate dehydrogenase phosphatase (PDP), activating:
- stimulated by:
- Ca2+, Mg2+
What is an anaplerotic reaction and what are the examples of anaplerotic reactions in the TCA cycle?
Under what conditions do they occur?
L4 S13 LO6
Fed conditions:
-carboxylation of pyruvate to form acetyl CoA
Starved conditions:
- degradation of amino acids
- glutamate (Gln/Pro/His/Arg) enters as α-ketoglutarate
- propionyl CoA (intermediate for Thr/Met/Ile/Val) enters as succinyl CoA
- Phe/Tyr/Asp enter as fumarate
What citric acid cycle intermediates are used in anabolic functions and what are they used to generate?
L4 S14 LO6
Citrate:
-fatty acids/isoprenoids
Malate:
-glucose
α-ketoglutarate:
-certain AAs
Oxaloacetate:
-certain AAs
What is the significance of thymaine (vitamin B1) deficiency?
L4 LO4
Panini pg. 129, blue box
Thyamine is used to make TPP which is a cofactor in PDC and α-ketoglutarate dehydrogenase.
Nutritional deficiency is called Beriberi.
Wernicker-Korsakoff syndrome is a deficiecny due to poor absorption as a result of alcoholism.
What effect does citrate have on enzyme function?
L4 LO5
Panini pg. 130, orange box
Citrate is indiciative of an ATP-rich state.
It inhibits PFK to prevent further production of ATP.
It activates acetyl CoA carboxylase promoting storage of energy as fat.
What are the differnt methods of uncoupling and what are examples?
L4 S35 LO11
Membrane damage:
- AraC
- AZT
Proton carriers:
- DNP
- Aspirin
Proton channels:
-UCP-1
How does NADH enter the mitochondria?
L4 S38-40 LO12
NADH is not permeable so it must be converted, transported, and then regenerated via…
Malate-aspartate shuttle (heart, liver, and kidneys)
Glycerophosphate shuttle (muslce and brain)
What is Luft’s disease?
L4 S42-44 LO13
Excessive uncoupling of oxidative phosphorylation resulting in dramatically increased BMR. First dx mitochondrial disease.
Acetyl CoA supply is obtained from the degradation of what nutrients?
1) Carbs:
Degraded from glucose which is oxidized to pyruvate
- Pyruvate is then decarboxylated via the PDH complex to generate A-CoA
Lipids:
-Fats stored as triacylglycerols in adipose tissues are degraded into FA which are then broken down into Acetyl-CoA units via Beta oxidation
Proteins:
- 7 ketogenic AA that are broken from proteins can be converted to acetyl Coa
- These include threonine, tryptophan, tyrosine, lysine, leucine, phenylalanine, and isoleucine
How is PDC regulated?
- (PDP) Pyruvate dehydrogenase phosphatase (activates the enzyme)
- (PDK) Pyruvate dehydrogenase kinase (deactivated the enzyme)
What stimulates and what inhibits PDC?
STIMULATED: -Ca2+ (cadiac muscle will inhibit PDK), Mg 2+ (alloesteically activates PDP)
- ADP, CoA, NAD, Pyruvate (ihibits PDK)
- Insulin (in adipose tissues) and cataholimines in cardiac muscles
INHIBITED: Acetyl CoA and NADH (activate PDK allosterically)
A-CoA, ATP, NADH all activate PDK
Aresnite (inhibits PDC by binding to lipoic acid in E2)
Explain PDC inhibition during fasting conditions:
Inhibition is favored under fasting conditions where levels of A-COA and NADH are high due to the oxidation of FA
neonatal lactic acidosis is caused by what?
Pyruvate dehydrogenase deficiency
- defects in PDC (particularly in E1)
- can be treated by vitamin B1 (B1 cofactor), Lipoic acid and biotin intake
- Treatment also includes dichloroacetate which is an inhibitor of PDK and serves to activate PDC
- ketogenic diet is recomended
Describe the effect of arsenite on lipoic acid
- lipoic acid subunit (E2) of the PDC is modified by arsenite (derivate of arsenic)
- Arsenite links to lipoic acids two sulfhydryl groups
- It is a suicide ihibitor that binds to thoil groups irreversibly and limits the avalibility of lipoic acid
- It will affect the PDC and other enzymes that use lipoic acid as a coenzyme (including alpha ketoglutarate dehydrogenase)
-Arsenic is a slow poison because it takes time to affect enough enzymes to become lethal
It builds up in the body and can be detected in hair
Explain Beriberi and Wernicke-Korsakoff syndrome
- nutritional deficiency condition in which the body does not have sufficient thiamine (vitamin B1)
- These conditions are diagnosed by measuring blood levels of thiamine.
- Treatment usually involves thiamine supplementation together with other water-soluble vitamins.
- Thiamine deficiency is commonly seen in alcoholics due to their poor nutrition and because ethanol tends to inhibit the absorption of thiamine.
- Thiamine deficiency in chronic alcoholics is termed Wernicke-Korsakoff syndrome.
What will a thiamine defiecny lead to?
Thiamine deficiency leads to increased blood levels of pyruvate and a-ketoglutarate due to impaired action of PDC and alpha ketoglutarate dehydrogenase, which requires thiamine pyrophosphate as an essential cofactor.
Which enzyme is found in the inner mitochodiral membrane?
succinate dehydrogenase
What does one turn of the TCA cycle make?
-1 oxaloacetate
-2 co2
-3 NADH (3 ATP/1 NADH)
- 1 fadh2 (2ATP/ 1NADH)
1 GTP (1ATP/ 1GTP)
Citrate regulates what?
- PFK1 in glycolysis
- citrate serves as an allosteric inhibitor of PFK- 1, the rate-limiting enzyme of glycolysis, to limit further catabolism of glucose.
- Citrate is also an allosteric activator of acetyl CoA carboxylase, which converts acetyl CoA to malonyl CoA in the rate-limiting first step of fatty acid biosynthesis.
Succinyl CoA in heme synthesis
- made from the TCA but used for heme synthesis
- the condensation of succinyl CoA and the AA glycine are decarboxylated to make gama aminovulinic acid as the first step in heme biosynthesis
- This reaction is catalyzed by ALA synthase the RLS of heme biosynthesis and requires pyridoxal phosphate as a cofactor
Rat Poison inhibition
- FLUOROACETATE IS A RAT POISON that inhibits the TCA cycle
- It reacts with COA to form fluoroacetyl coa which condenses with oxaloacetate to produce fluorocitrate
- Fluorocitrate is an analogue of citrate and is a competitive inhibitor of aconitase
- Thus inhibition of aconitase leads to the accumulation of citrate which inhibits citrate synthase
- Fluorocitrate, like citrate, is an allosteric inhibitor of pfk 1 and acts to inhibit glycolysis
Anaplerotic reactions and anabolic functions of the TCA cycle
o Degradation of AA: Replenish oxaloacetate, alpha ketoglutarate, succinyl CoA
and fumarate
Carboxylation of Pyruvate: Replenishes oxaloacetate via pyruvate carboxylase
The enzyme needs biotin for its activity and is stimulated by A-CoA
- Occurs during glucose deprived conditions in which the A-COA supply is high
- The resulting oxaoacetate will be used to synthesize glucose via gluconeogenesis
What does OAA need to function?
Biotin (V7) a coenzyme
Pyruvate Carboxylase Deficiency:
- Disruption of the gene causes more pyruvate to be converted to lactic acid than oxaloacetate
- Lactic acid will accumulate in the blood
- The symptoms of the condition appear after birth and include seizures, muscle weakness, uncontrolled muscle movement
- It is an autosomal recessive pattern and more prevelant in the algokian indian tribes in canada
- Prevlance is 1/250,000
Mitochondria matrix is the site for what?
Mitochondrial matrix: has pH higher than intermembrane space and serves the site for beta oxidation of FA, pyruvate dehydrogenase activity, TCA, ketone body synthesis, some reactions of the urea cycle, and heme biosynthesis, and a reservoir for ca2
Outer mitochondrial membrane
Permeable to small molecules due to the presence of protein channels called porins
Ox phos is mediated by the________.
Define the latter.
- Respiratory Chain
- a collection of inner mitochondrial membrane proteins called complexes I-V, cytochrome c, and lipophilic molecules co enzyme Q (ubiquinone)
electron only transfer
eletrons are transferred between two metal ions
Reducing Equivalent Transfer
involves transfer of a proton plus an electron.
Lower redox potential means ___.
Higher redox potential means ___.
reducing agent (GETS OXIDIZED)
oxidizing agent (gets reduced)
Ferredoxins
Ferredoxins are small proteins that serve as carriers of electrons in mitochondrial cytochrome P-450 systems.
- They contain clusters of iron and sulfur.
- Reduction and oxidation of iron between Fe3+ and Fe2+ states allow transfer of electrons between members of the ferredoxin family.
Mechanism of oxidative phosphorylation: THREE GOALS
1) To transfer electrons from NADH and FADH2 to O2
2) To generate a proton gradient
3) To make ATP
cytosine arabinoside (ARAC) / AZIDOTHYMIDINE (AZT)
This is a ETC uncoupler which is a membrane damaging agents:
- REDUCE THE HYDROGEN GRADIENT!!!
- These include cytosine arabinoside (ARAC): Used to treat cancer
-AZIDOTHYMIDINE (AZT)
Used to treat HIV
DNP (2,4-dinitrophenol)
Type of ETC uncoupler. Moblie proton carriers
-Certain lipid soluble substances bind to and transport protons through inner membranes.
unionized lipid soluble form of aspirin
mobile proton carrier
Themogenesis in Brown Adipose tissues:
Unlike white adipose, it is rich in mitochondria and has a high expression of UCP-1 (uncoupling protein-1, thermogenin) in the inner mitochondrial membranes.
- uncouples the electron transport from ATP synthase by providing a pathway for protons to leak across the inner mitochondrial membrane back into the matrix.
- The energy stored in the proton gradient is dissipated as heat rather than used for the production of ATP.
- -Norepinephrine is released by postganglionic sympathetic nerves to activate b-adrenergic receptors on the surface of brown adipose cells.
- As a result, a cytosolic lipase is stimulated to break down triglycerides into fatty acids.
- The fatty acids activate UCP- 1 and stimulate H+ transport to release heat
- It is thought that a long-chain fatty acid anion binds to UCP-1 in a non-dissociable fashion and helps in the symport of a proton into the matrix.
Chronic norepinephrine signaling also upregulates gene expression of UCP-1, leading to increased mitochondrial biogenesis.
Rotenone
- potent inhibitor of NADH dehydrogenase (complex 1) as it prevents eletron transfer from FE-S centers to ubiquinone
- ihibiton may be over come by menadione (vit k3) which alows eletrons to bypass the site of rotenone block
- acute poisoning treatment include: washing skin, eyes, gastric lavage, IV administration of glucose and menadione
- chronic rotenone poisioning has in some cases been linked to parkinsons
Cyanide Poisioning
- CN binds to oxidized (ferric) form of iorn in the heme of cytochrome a3 component of complex 4 and prevents o2 reduction (terminal step of ETC)
- -If caught early nitrites can convert Fe2+ to Fe3+ resulting in the formation of methemoglobin which competes for the binding of CN to complex 4
Azide
inhibits complex 4 by binding to fe3+
Cyaninde vs Carbon Monoxide action
- both CO and NO compete with O2 for binding to reduced heme a3 in complex 4
- CN, N3- and Hydrogen Sulfide (H2S) compete to bind to oxidized form of heme a3 (fe3+)
- thus CO acts as a competitive inhibitor (decreases Vmax)
- CN and H2S acts as a non competitive inhibitor (decreases Vmax)
Asprin Overdose
- also known as acetyl salicylate
- acute asprin poisoning at does 150 mgs/kg paradoxically causes hyperthermia
- salicylate uncouples ox phos by disrupting the protein gradient and causes dissipation of heat
- also stimulates brain respiratory center and causes hyperventilation
Antimycin
Inhibits complex 3
Malonate
inhibits complex 2
What are AZT and Ara C used for
- used in the antiretroviral therapy
- The drugs inhibit mitochondrial dns poly, and deplete mitochondrial DNA which causes a decrease in locally produced components of respiratory chain especially complex
- when complex 1 is inhibited, atp production is halted and causes an over utilization of complex 2 resulting in elevated levels of ROS and further damaging other cellular systems.
Hypoxia and starvation
- oxygen starvation also causes cells to depend on glycolysis for energy production leading to lactic acidosis
- lower pH in the mitochodrial matrix causes dimerization and activation of small ihibitory protien IF1 that binds to ATP synthase and prevents it from active to hydrolyze ATP. This helps preserve ATP
cytochrome c and apoptosis
- Cytochrome C is a protein that functions in complex 3 and 4
- apoptosis is programmed cell death (occurs for many reasons such as: DNA damage, normal development, growth factor deprivation.
- apoptotic stimuli initiate the process by opening the mitochondrial permeability transition pore complex which causes a release of cytochrome
- when cytochrome c is in the cytosol it serves as a miiochemical marker that the cell is undergoing apoptosis
Malate aspartate shuttle system
operated in heart, liver and kidneys
-point is to transfer an NADH eletron in the matrix from the cytosol
Glycerophosphate shuttle:
operates in skeletal muscle and brain
-Nadh eletron is trasnfered to a FADH on complex 2
Antiports
- phosphate/ OH antiport (driving force is pH gradient)
- phosphate malate antiport (phosphate in malate out)
- ADP/ATP antiport (driving force is membrane potential and pH)
- pyruvate Oh antiport (OH out and pyruvate in)
Creatine Kinase: marker for MI
- creatine kinases phosphorylate creatine to generate the high energy compund phosphocreatine which serves as an energy storage reserve in resring muscles
- when needed phosphocreatine donates its phosphate group for phos of ADP to ATP to help support muscle contractions
- the form of ck that is found in muscle and brain tissue is CK-MB and is used as a marker for MI because damage to heart mucles result in high levels of serum ck-mb within the first 4-8 levels of MI
- CK-MB also can go up if there is damage to skeletal muscle also, thus conformation is needed with other cardiac markers such as troponin T and glycogen phosporylase BB
What is mitrocondrial fission?
essential event in proliferating cells (creates new organneles and facilitates quality control)
Mitochondrial fusion
collions by movment of organelles may lead to fusion
-this is for an effort to maintain health mitochondrial network and rescuing of less functional mitochondria
2-Oxoglutaric acid aciduria
LOOK UP
Fumerase defienciey
Ubiqinone radicals
dot Q- is a intermediate when transfering eletrons to complex 1 to ubiqninone and transfering reduced ubiqnione to complex 3
- it can pass its radical to O2- to make a super radical
- H2o2, Oh- radiacl and o2 radiacl are ROS in mmitochondria
- cause damage to membrane and lipids
