Oxidative Phosphorylation and Gluconeogenesis Flashcards
Describe the mechanisms that are responsible for the generation of the energy that is used to drive oxidative phosphorylation
NADH and FADH2 are energy rich coenzyme molecules that are produced from various metabolic processes. NADH and FADH2 pass their electrons down a series of protein complexes in the inner mitochondrial membrane and ultimately to oxygen. This flow of electrons is coupled to pumping of protons across the inner membrane and into the inter membrane space. Energy derived from protons moving down the pH gradient and into the mitochondrial matrix is used to create ATP.
Describe the components of the electron transport chain and their location within the mitochondria.
The electron transport chain is comprised of a series of protein complexes on the inner membrane of the mitochondria.
Complexes I-IV plus Cytochrome C and Coenzyme Q (a quinone, not a protein) are actually involved in transfer of e- and the pumping of protons into the inter membrane space.
Complex V - “ATP synthase” does not handle e- but creates ATP from ADP and Pi via protons flowing down the pH gradient.
There is also a special carrier molecule for transporting ADP into the mitochondria and ATP out to the cytosol.
What are the substrates that are used in oxidative phosphorylation?
Nicotinamide Adenine Dinucleotide (NAD+) and Flavin Adenine Dinucleotide (FAD). These coenzymes are reduced by metabolic intermediates to NADH and FADH2. Their reduced forms are able to transfer the electrons to the proteins of the electron transport chain.
NADH = ~2.5ATP
FADH2 = ~1.5ATP
Difference is due to the fact that NADH interacts with complex I but FADH2 starts at complex III.
What are the consequences of defects in electron transport?
Defects in electron transport result in the inability to generate ATP. Tissues with the highest energy demands will be most effected. These tissues include CNS, skeletal muscle, cardiac muscle, kidney, and liver. Without ATP, tissues do not have the energy required for normal functions and problems like neuropathies and myopathies result. Defects in oxidative phosphorylation are most often the result of mutations in mitochondrial DNA.
List inhibitors of electron transport
Oligomycin inhibits the synthesis of ATP, resulting in the buildup of NADH and FADH2 which reduce flux through the TCA cycle. Cells must then rely on glycolysis which is inefficient and causes an increase in serum lactic acid.
Carbon Monoxide poisoning results in the inability of blood to deliver oxygen to tissues. This inhibits the function of the electron transport chain while maintaining high pO2 concentrations.
Uncoupling proteins allow the proton gradient across the inner membrane to dissipate without coupling to ATP generation and results in loss of the chemical energy as heat. This mechanism is used by brown adipose tissue to generate heat to keep the body warm in cold conditions.
What are the clinical consequences of errors in mitochondrial function?
Inborn errors of mitochondrial function commonly result in dysfunction of neural tissues or skeletal muscle because these tissues require a great deal of energy for normal functioning. They can present as retinal dysfunction, encephalopathy or myopathy, especially following exercise.
What is the role of PGC1 in mitochondrial biogenesis?
PGC1 appears to be one of the key molecular mediators of mitochondrial proliferation. It is thought that increased proliferation of mitochondria would limit the adverse effects of overfeeding and possibly ameliorate or cure metabolic diseases.
What is the metabolic role of gluconeogenesis?
Gluconeogenesis is the process that creates glucose from non-carbohydrate precursors during times of fasting, vigorous exercise, low-carbohydrate diets, or physiologic stress. It is particularly important in tissues that require glucose as their sole source of energy (neurons, RBCs, renal medulla, sperm, embryonic tissues). Glycogen stores can maintain a person for only 1-2 days. Gluconeogenesis occurs primarily in the liver, but may also occur in the kidney in times of prolonged starvation.
What are the major precursors for gluconeogenesis?
LACTATE: Generated in RBCs and skeletal muscle when the rate of glycolysis exceeds the rate of the TCA and ETC. Lactate then moves through the blood to the liver where it enters gluconeogensis in hepatocyte cytoplasm.
AMINO ACIDS: Provide carbon skeletons to the TCA cycle and progress to gluconeogenesis via oxaloacetate. Most enter as succinate or fumarate, glutamine enters as a-ketoglutarate, alanine is directly converted to pyruvate.
GLYCEROL: Released during the hydrolysis of tryglycerides in adipose tissue. Travels through the blood to the liver and enters gluconeogenesis as dihydroxyacetone phosphate, an intermediate of glycolysis.
What is the Cori cycle?
The Cori cycle involves Glycolysis resulting in lactate production in RBCs (or other cells), the transfer of that lactate through the blood stream to the liver, reuptake of the lactate into hepatocytes, and conversion of the lactate into pyruvate to enter gluconeogenesis in the hepatocytes. The new glucose is then available for use in other tissues or storage as glycogen.
What is the primary mechanism responsible for coordination of glycolysis or gluconeogenesis?
Glucagon concentration and the phosphorylation or dephosphorylation of the PFK2/FBP2 bifunctional enzyme.
In its dephosphorylated state, the PFK2 enzyme is active, creating F-2,6,-BP, a potent stimulator of PFK1/driver of glycolysis and inhibitor of FBP1/driver of gluconeogenesis.
In its phosphorylated state, the FBP2 enzyme is active, destroying F-2,6-BP, inhibiting PFK1 and glycolysis, and stimulating gluconeogensis by disinhibition of FBP1.
Glucagon stimulates the phosphorylation of PFK2/FBP2 via stimulation of adenylyl cyclase -> increased cAMP -> activation of PKA -> directly phosphorylated PFK2/FBP2. Hence, high glucagon leads to gluconeogenesis, low glucagon leads to glycolysis.
What are the key steps in gluconeogenesis?
Gluconeogenesis must overcome the irreversible steps of glycolysis, thus its key steps are the same as the key steps of glycolysis, but in reverse order:
1 - Pyruvate -> Phosphoenol Pyruvate (PEP) via oxaoloacetate
2 - Fructose-1,6-bisphosphate -> Fructose-6-phosphate
3 - Glucose-6-phosphate -> Glucose
How is the first key step of gluconeogenesis completed?
The first key step of gluconeogenesis is the conversion of pyruvate back into phosphoenol pyruvate. (Only important for precursors that enter as pyruvate!!) This is achieved through coordination of mitochondrial and cytoplasmic enzymes, so pyruvate is first imported into the mitochondria. There it is converted to oxaloacetate (4C) by PYRUVATE CARBOXYLASE and ATP. Coenzyme BIOTIN is required, thus biotin deficiency leads to build up of pyruvate and lactic acidosis. OAA must then be converted to MALATE (via malate dehydrogenase) to the exported from the mitochondria via MALATE/A-KETOGLUTARATE transporter, and reconverted to OAA in the cytosol (malate dehydrogenase again). OAA is then converted to phosphoenol pyruvate by PEPCK (carboxykinase) in the cytosol. Total reaction requires 1 ATP and 1 GTP.
Pyruvate -> OAA -> Malate -> OAA -> PEP
Mitochondrial enzymes: Pyruvate Carboxylase & Biotin, Malate Dehydrogenase
Cytosolic enzymes: Malate Dehydrogenase, PEPCK
Transporters: Pyruvate, Malate/a-ketoglutarate
Energy: 1 ATP, 1 GTP, produced from lipid TCA breakdown
How is the second key step of gluconeogenesis completed?
Conversion of Fructose-1,6-bisphosphate to F6P. Glucagon signaling increases the activity of adenylyl cyclase which increases cAMP concentrations, activating PKA, which phosphorylates PFK2/FBP2, leading to the activation of the FBPhosphatase enzyme. This converts F-2,6-BP to F6P, inhibits PFK1 activity, and stimulates FBP1 (fructose bisphosphatase 1, removes the 1-phosphate group). FBP1 catalyses conversion of F-1,6-BP to F6P.
Reaction: F-1,6-BP -> F6P
Enzymes: Fructose-1,6-bisphosphatase (FBP1), Fructose-2,6-bisphosphatase (FBP2)
Control Molecules: Glucagon, F-2,6-BP
How is the third key step of gluconeogenesis completed?
Conversion of G6P to Glucose. This reaction bypasses the hexokinase reaction and is accomplished by glucose-6-phosphatase. G6-phosphatase is located in the membrane of the ER in hepatocytes and kidney cells. G6P is transported into the ER, converted to Glucose, and then exported out of the cell into the blood. Tissues that lack this enzyme (eg skeletal muscle) cannot export glucose.
CLINICAL CORRELATE: Von Gierke’s disease (AR), is a deficiency of G6Phosphatase in the liver. Glycogen remains normal, but patients have severe fasting hypoglycemia, ketosis, lactic acidosis, enlarged liver, and kidneys.
Reaction: G6P -> Glucose
Enzyme: Glucose-6-phosphatase
