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Where does the citric acid (TCA) cycle occur?
Mitochondria
What does the citric acid cycle produce per 1 molecule of Acetyl-CoA?
3 NADH
1 FADH2
1 GTP
2 CO2
What is the committed step of the citric acid cycle?
Formation of citrate
(Acetyl CoA undergoes condensation rxn with oxaloacetate to form citrate)
Where do the molecules produced in the citric acid cycle go?
Electron Transport Chain (ETC)
What can Acetyl CoA be formed from?
Pyruvate
How do Acetyl CoA form?
Pyruvate is produced by glycolysis in the cytoplasm. It is transported to the mitochondria where it undergoes oxidative decarboxylation and is converted to Acetyl CoA via the enzyme Pyruvate Dehydrogenase. This conversion is also accompanied by the reduction of NAD+ to NADH.
The Citric Acid Cycle
What does a large negative delta G indicate?
Tells us the reaction is highly exergonic and highly favorable. This will indicate there needs to be regulation of some sort at these given steps.
Which reactions of the TCA cycle produce NADH?
Reactions 3, 4, and 8
(isocitrate --> alpha-ketoglutarate) (alpha-ketoglutarate --> succinyl CoA) (malate --> oxaloacetate)
NADH is used for ETC
Which reaction of the TCA cycle produce FADH2?
Reaction 6
(succinate --> fumarate)
Which reactions of the TCA cycle use H2O as a reactant?
Reactions 1 and 7
(acetyl CoA + oxaloacetate --> citrate) (fumarate --> malate)
Which reaction of the TCA cycle produces GTP?
Reaction 5 produces GTP
(succinyl CoA --> succinate)
How does the oxidation of 1 glucose molecule into 2 pyruvate molecules form 8 ATP equivalents?
1 glucose is oxidized to 2 pyruvate which makes 2 ATP. 2 NADH makes 6 ATP from Glycolysis (8 total).
How does the conversion of 2 pyruvate into 2 Acetyl CoA form 6 ATP equivalents?
1 molecule of NAD+ is reduced to NADH when pyruvate is converted into Acetyl CoA. 1 NAD+ gives 3 ATP, since there are 2 NAD+ it makes six ATP total when oxidized.
How does the oxidation of 2 Acetyl CoA in the TCA cycle produce 24 ATP equivalents?
12 ATP equivalents are formed from the complete oxidation of one Acetyl-CoA. Since there are 2 pyruvate being converted to 2 Acetyl CoA, there will be 24 ATP total.
3 NADH = 9 ATP x 2 = 18 ATP
1 FADH2 = 2 ATP x 2 = 4 ATP
1 GTP = 1 ATP x 2 = 2 ATP
Total: 24 ATP
How many total ATP equivalents are formed from the complete oxidation of one glucose molecule?
Up to 34
What are anaplerotic (filling up) reactions?
Reactions providing intermediates to the TCA cycle that are not oxaloacetate
What determines the rate of TCA?
Concentrations of oxaloacetate in the cell determine the rate of TCA
What are the 3 major anaplerotic reactions?
Pyruvate to Oxaloacetate via pyruvate carboxylase
PEP to oxaloacetate via PEP carboxylase
Pyruvate to Malate via malic enzyme
Why is PEP carboxykinase not anaplerotic?
It goes the wrong direction
What are the 3 reactions that are the focus of regulation in the TCA cycle?
Step 1: Oxaloacetate + Acetyl CoA to Citrate (catalyzed by citrate synthase)
Step 3: Isocitrate to a-ketoglutarate + NADH (catalyzed by isocitrate dehydrogenase)
Step 4: a-ketoglutarate to Succinyl-CoA (catalyzed by a-ketoglutarate dehydrogenase)
Why is TCA regulated at multiple points?
Too fast = energy wasted, buildup of cofactors/coenzymes becomes toxic
Too slow = not enough energy to function and produce intermediates...might die
What regulates Citrate Synthase?
ATP = inhibit
NADH = inhibit
Succinyl-CoA = inhibit
What regulates Isocitrate Dehydrogenase?
ATP = inhibit
ADP = activate
NAD+ = activate
What regulates alpha-ketoglutarate dehydrogenase?
NADH = inhibit (this step produces NADH so if there is enough...inhibit)
Succinyl-CoA = inhibit (this step produces Succinyl-CoA...doesn't need to produce more if enough already)
AMP = activate
What is the purpose of the pyruvate dehydrogenase complex?
Converts pyruvate to Acetyl CoA and then determines whether this molecule will enter into the TCA cycle or continue on to Fatty Acid Synthesis
How is pyruvate dehydrogenase (PDH) regulated?
Covalently via Phosphorylation
Allosterically
phosphorylated = inactive
de-phosphorylated = active
products inhibit enzyme
substrates promote enzyme
High NADH/Acetyl-CoA concentrations allosterically activate PDH to phosphorylate serine 203, 264, 271 on alpha subunit of PDH (blocks initial PDH rxn)
PDH reactivated by Ca2+ dependent enzyme PDH phosphatase (hydrolyzes phosphoserines)
Low NADH or high NAD+ or low Acetyl-CoA concentrations activate PDH phosphatase
Insulin/Ca2+ ions activate PDH and pyruvate blocks phosphorylation
Describe the basics of the other 3 layers of regulation associated with the pyruvate dehydrogenase complex
Acetyl CoA blocks dihydrolipoyl transacetylase
NADH inhibits dihyrolipoyl dehydrogenase
AMP activates pyruvate dehydrogenase
GTP inhibits pyruvate dehydrogenase
Understand how Lysine Acetylation is a regulatory mechanism that all enzymes under the TCA cycle are held under
Acetylation inhibits TCA activity
SIRT3 is mitochondrial NAD+ dependent deacetylase responsible for deacylation and activation of TCA enzymes
Discuss SIRT3 expression in the body
SIRT3 expression increases with exercise
The more you exercise, the more energy you need (SIRT3 is produced to keep TCA actively producing energy)
SIRT3 production is linked to longevity in humans
What is the goal of the Electron Transport Chain?
To use oxidative phosphorylation to convert ADP to ATP
NADH/FADH2 dependent ATP synthesis
NADH/FADH2 are oxidized
How much ATP does 1 glucose yield in the Electron Transport Chain?
34 ATP
Describe key features of the mitochondria
Outer membrane is porous/permeable to ions and small molecules
Inner membrane is impermeable and transport requires proteins (all enzymes/proteins required for oxidative phosphorylation are in the inner membrane)
Inner membrane contains enzymes adenylate kinase and nucleoside-diphosphate kinase (play role in energy metabolism)
Mitochondrial matrix contains oxidative enzymes, pyruvate dehydrogenase (PDH), and enzymes of TCA and fatty acid (beta) oxidation
Is the electron transport chain aerobic or anaerobic?
Aerobic
What are the 4 complexes involved in the ETC?
Complex I (NADH CoQ Reductase)
Complex II (Succinate-CoQ Reductase)
Complex III (CoQ-Cytochrome C Reductase)
Complex IV (Cytochrome C Oxidase)
Electron Transport Chain (ETC)
Does Complex I of the ETC pump protons? If so, how many?
4 protons are pumped for every NADH oxidized
Complex I catalyzes the oxidation of _________ and reduction of ____________ .
NADH (oxidized)
Ubiquinone (reduced)
What carrier carries electrons from Complex I to Complex III
Coenzyme Q (CoQ)
also called ubiquinone
2 electrons are passed from NADH --> FMNH2 --> Fe-S --> CoQ (ubiquinone)
Are protons pumped in Complex II of the ETC? If so, how many?
No protons pumped
Complex II of the ETC catalyzes the oxidation of _________ and reduction of __________ .
FADH2 (from TCA) oxidized
Ubiquinone (UQ) reduced to (UQH2)
What is succinate oxidized into in Complex II of the ETC?
Fumarate
Complex III of the ETC catalyzes oxidation of __________ and reduction of __________ .
1 UQH2 (oxidized)
2 Cytochrome C molecules (reduced)
Complex III accepts electrons from coenzyme QH2 that is generated from which complex(es)?
Complex II
What cycle is enacted in complex III?
Q Cycle
What is the Q cycle? What does it do?
Mechanism for coupling electron transfer from QH2 to cytochrome c found in Complex III
The iron (Fe) in the cytochrome proteins is located in a porphyrin-ring structure (like heme), and can exist in what two oxidation states?
Fe2+ and Fe3+
Are protons pumped in Complex IV? If so, how many?
4 protons are transported across the inner membrane of the mitochondria
Electrons combine with ________ and _______ to form 2 molecules of water
Cytochrome C Oxidase accepts electrons from reduced Cytochrome C (complex III) and used them to reduce molecular oxygen (O2) to water (H2O)
Explain proton/electron movement in Complex IV of the ETC
For every 4 electrons passing through Complex IV, 8 protons are removed from the matrix side of the membrane
(4 protons are transferred to intermembrane space, other 4 protons end up in water molecules)
How is the proton motor force established?
The differences the protons make both in concentration and electrostatic gradients across the membrane can be related to free energy
Is the export of protons favorable or unfavorable?
Unfavorable
(export of protons has has a positive free energy change)
By default, is the pulling of protons across the membrane favorable or unfavorable?
Favorable
(Delta G = -23.3 kJ/mol thus it IS favorable)
What is the ATP Synthase?
The enzyme complex that carries out ATP synthesis in the mitochondria
What is another name for ATP Synthase?
F1-F0-ATPase
What are the two parts that make up the ATP Synthase?
F1 and F0
What is the F1 complex of ATP Synthase responsible for?
F1 performs the ATP synthesis activity
F1 protrudes into the matrix environment
Contains 5 subunits (a,b,g,d,e)
F1 complex's 'b' subunit is the location of synthesis activity
c,g,e rotate as protons flow through (rotor)
What is the F0 complex of ATP Synthase responsible for?
F0 is designed for the flow of protons (embedded in membrane)
rotates counterclockwise
F0 has 3 subunits (a,b,c)
Has transmembrane pore from the intermembrane space to the matrix
Describe the 3 conformations the ATP Synthase goes through while rotating
OPEN
low affinity for ATP
not catalytically active
LOOSE
loosely binds ATP, ADP, phosphate ions
not catalytically active
TIGHT
tightly binds ATP, ADP, phosphate ions
catalytically active
Which direction does the c-subunit of F0 of ATP Synthase rotate? Why?
Counterclockwise because it is energetically favorable and there are no repulsive interactions between the amino acids that make up the subunit and the protons being pumped from the highly concentrated intermembrane space to the less concentrated matrix
How many protons (H+) are required to make 1 ATP?
3 protons
How many protons (H+) are made from 1 NADH?
10 protons
How many ATP can form from 1 NADH?
1 NADH produces 10 protons
If 3 protons make up 1 ATP, then
10/3 = 3.33 ATP
How does Rotenone inhibit the ETC?
Inhibits NADH-UQ Reductase in Complex I
How do Barbiturates inhibit the ETC?
Inhibit NADH-UQ Reductase in Complex I
How does Demerol inhibit the ETC?
Inhibits NADH-UQ Reductase in Complex I
(Demerol is a pain killer)
How does Cyanide inhibit the ETC?
Binds tightly to the ferric form of Cytochrome A in Complex IV
How does Azide inhibit the ETC?
Binds tightly to the ferric form of Cytochrome A in Complex IV
How does Carbon Monoxide inhibit the ETC?
Binds tightly to the ferric form of Cytochrome A in Complex IV
How does Oligomycin inhibit the ETC?
Blocks movement of protons through the F0 complex of ATP Synthase
What do uncouplers do?
Dissipate proton gradient across inner mitochondrial membrane
Combine with cytosolic protons and carry them back into the matrix
(Uncouplers produce heat/release energy from the uncoupled flow of protons through the membrane...a mechanism by which an organism can warm itself up when needed)
(ATP Synthase needs proton gradient to fxn...uncouplers undo gradient)
What are physical properties of uncouplers?
They are hydrophobic molecules with functional groups that can be protonated or deprotonated
What are 2 examples of uncouplers?
2,4-Dinitrophenol and Dicumarol
What is ATP-ADP Translocase?
An enzyme that moves ATP from the mitochondria to other parts of the cell for use and brings ADP back into the mitochondria for "recharging"
Describe how ATP-ADP Translocase works?
The enzyme transfers 1 ADP into the mitochondria for every 1 ATP that is transferred out
(the charge difference (1-) between ATP (4-) and ADP (3-) as well as the concentration gradient makes these transfers spontaneous)
(an additional proton must be used to neutralize (1-) charge, thus total cost of protons to 1 ATP production is 4:1)
What is the P/O Ratio? (Phosphate/Oxygen Ratio)
The ratio between the number of molecules of ATP formed in oxidative phosphorylation for every 2 electrons flowing through a defined segment of the ETC
What does the P/O Ratio assume?
Assume 10 protons out of matrix for every 2 electrons passing through ETC, and 4 protons transported from cytosol into matrix
(~ 2.5 molecules of ATP per 2 electrons flowing)
List the steps of the Glycerophosphate shuttle
Electrons in NADH are transferred to DHAP to produce G-3-P and NAD+
G-3-P transfers its electrons directly to FAD in the inner mitochondrial membrane producing FADH2
FADH2 proceeds through the ETC
How much ATP is created per glucose molecule through the Glycerophosphate shuttle?
32 ATP
Is the malate aspartate shuttle reversible or irreversible?
Reversible
List the steps of the malate aspartate shuttle
NADH oxidized in cytosol by reducing Oxaloacetate to Malate
Electrons brought into matrix as Malate is transported into matrix by a-ketoglutarate-malate transporter
Malate converted back into Oxaloacetate by Malate Dehydrogenase with concurrent reduction of NAD+ to NADH
Oxaloacetate is transaminated to Aspartate by the Aspartate Aminotransferase
Aspartate and Glutamate swap sides of membrane via Aspartate-Glutamate Carrier
(no charge difference, thus requires no additional protons...more efficient than glycerophosphate shuttle)
How many ATP are made per glucose if the malate-aspartate shuttle is used?
34 ATP
What is the goal of gluconeogenesis?
To create glucose from non-carbohydrate precursors
What are the non-carbohydrate precursors of gluconeogenesis?
Pyruvate, all amino acids (except lysine/leucine), lactic acid, glycerol, and any TCA intermediate
How many steps of Gluconeogenesis are retained from Glycolysis? Which ones?
7 steps retained
(steps 2 and 4-9)
What are the 3 steps of Glycolysis replaced in Gluconeogenesis?
Steps 1, 3, and 10 (pyruvate carboxylase, PEP carboxylase, fructose-1,6-bisphosphatase, glucose 6 phosphatase)
What do the new enzymes of gluconeogenesis replace in glycolysis?
Pyruvate carboxylase and PEP carboxykinase replace pyruvate kinase
Fructose-1,6-bisphosphatase replaces phosphofructokinase
Glucose-6-phosphatase replaces hexokinase
(These new rxns provide for spontaneous pathway in reverse direction of glycolysis...also provides new mechanism of regulation)
Where does gluconeogenesis occur?
Mainly in the liver and kidneys
What does Pyruvate Carboxylase do and how is it regulated?
Converts Pyruvate to Oxaloacetate (uses ATP and CO2)
Allosterically activated by Acetyl-CoA (body puts oxaloacetate and pyruvate into gluconeogenesis)
Acetyl CoA levels low = body pushes pyruvate and oxaloacetate into TCA and glycolysis
What does PEP carboxykinase do?
Converts oxaloacetate to PEP (uses GTP; makes CO2)
What does Fructose-1,6-bisphosphatase do?
Hydrolyzes fructose-1,6-bisphosphate to fructose-6-phosphate
What is the reaction for pyruvate carboxylase?
pyruvate + bicarbonate + ATP --> oxaloacetate + ADP + Pi
This enzyme is Biotin dependent (bicarbonate is the biotin)
What regulates Fructose-1,6-bisphosphatase?
Citrate stimulates
Fructose-2,6-bisphosphate inhibits
AMP inhibits
What is the reaction for fructose-1,6-bisphosphatase?
F-1,6-BP + H2O --> F-6-P + Pi
What does Glucose-6-phosphatase do?
Converts G-6-P to Glucose in the Endoplasmic Reticulum
Explain the Glucose-6-Phosphatase system
The glucose-6-phosphatase system includes phosphatase itself and 3 transport proteins (T1, T2, T3)
T1 takes G-6-P into the ER where it is hydrolyzed by the phosphatase
T2 and T3 export glucose and Pi, respectively, to the cytosol
GLUT2 exports glucose to the circulation
Explain 2 reasons why gluconeogenesis is not the mere reversal of glycolysis
If the processes were reversible there would be no room for regulation which is absolutely necessary (glycolysis on, gluconeogenesis off...vice versa)
Glycolysis has delta G of -74 kJ/mol...if gluconeogenesis were simply the reverse it would have a positive delta G which would make the reaction impossible
Why does gluconeogenesis only occur in the liver and kidneys?
Brain and muscles lack enzymes to allow gluconeogenesis to occur
Explain the Cori Cycle
Lactate is produced in the muscle during exercise. Lactate buildup causes cramping due to lack of oxygen
NADH reoxidized during reduction of pyruvate to lactate
Lactate returns to liver where it can be reoxidized to pyruvate by lactate dehydrogenase
Liver produces/provides glucose to muscle for exercise and then reprocesses lactate into new glucose
What does it mean that gluconeogenesis and glycolysis are under reciprocal control?
Glycolysis is inhibited when gluconeogenesis is running and vice versa
Note
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