FINAL STUDY GUIDE
THINGS TO KNOW:
Digestive enzymes (cut food up)
Synthase/Synthetase (combine two molecules to make a single product)
Kinase/Phosphatase (add/subtract phosphate)Isomerase/Mutase/Epimerase (etc) (rearrange molecule – reactant and product have same number of atoms)
Reductase/Oxidase (reduce or oxidize product)
Carboxylase (add or remove carbon dioxide)
Hydrogenase/Dehydrogenase (add or remove H2 in the form of H+ and H-)
Hydratase/Dehydratase (add or remove H2O; e.g. enolase)
Transaminase (transfer amine group)
Energy
Three types of molecules that are digested for energy?
Fats —> Fatty Acids
Carbohydrates —> glucose
Protein —> amino acids
What are some enzymes that break down food in the stomach and small intestine?
Protein is broken down by protease into oligopeptide
Carbs are broken down by amylase into maltose / dextrins
Fats are broken down by lipase into fatty acids
They are all broken down by hydration reactions
What are the forms of proteins and fat that can be passed from the lumen into a mucosal (intestinal cell)?
Amino acids (protein): The simplest form of proteins, absorbed directly into the bloodstream.
Tripeptides and Dipeptides (amino acids)
Fatty acids and monoglycerides: The products of fat digestion, which can easily diffuse across the intestinal cell membranes.
Define: metabolism, anabolism, catabolism
Metabolism: Molecule transformed into another molecule through enzymatic reactions
Anabolism: Building of molecules, uses energy
Catabolism: Breakdown of molecules, releases energy
What is the rationale for energy production upon hydrolysis of ATP gamma phosphate?
The hydrolysis of ATP gamma phosphate releases energy because it involves the breaking of a high-energy bond, resulting in ADP and inorganic phosphate (Pi), which creates a more stable state and drives various cellular processes.
Gamma phosphate: Third phosphate group in ATP, attaches to betaphosphate and is released during hydrolysis. Release energy.
How do relevant resonance structures for ATP/Pi affect reactivity?
ATP: Three phosphate groups (alpha, beta, gamma) that are negatively charged and repulse each other - leading to instability, more reactive. Hydrolysis is exergonic!
Pi: Inorganic phosphate group created as byproduct of ATP hydrolysis.
What chemical features make ADP/Pi more stable than ATP?
ADP: Product of ATP hydrolysis, only two phosphate groups (alpha, beta) that are less repulsed by each other, resulting in greater stability. Additionally, the removal of the gamma phosphate reduces the overall energy of the molecule, making it less reactive compared to ATP.
Pi: The inorganic phosphate group (Pi) is more stable due to its resonance stabilization, which allows for delocalization of negative charge, as well as its ability to form hydrogen bonds with surrounding water molecules, further stabilizing the structure.
Glycolysis/Gluconeogenesis
1. How is glucose trapped within the cell?
Glucose is too big to just pass through the cell membrane easily. So, once it enters the cell, it's converted into glucose-6-phosphate by an enzyme called hexokinase. This conversion “traps” glucose inside the cell because glucose-6-phosphate can't easily leave the cell, unlike glucose itself.
2. What is hexokinase's function?
Hexokinase is an enzyme that adds a phosphate group to glucose, turning it into glucose-6-phosphate. This is the first step in trapping glucose inside the cell and also starts the process of turning glucose into energy.
3. The structures, full names, and enzymes of the glycolysis pathway:
Glycolysis is the process where one molecule of glucose is broken down into two molecules of pyruvate, and energy is released. Here are the steps with the full names of the enzymes involved:
Glucose → Glucose-6-phosphate (by Hexokinase)
Glucose-6-phosphate → Fructose-6-phosphate (by Phosphoglucose isomerase)
Fructose-6-phosphate → Fructose-1,6-bisphosphate (by Phosphofructokinase-1, or PFK-1)
Fructose-1,6-bisphosphate → Glyceraldehyde-3-phosphate (G3P) + Dihydroxyacetone phosphate (DHAP) (by Aldolase)
DHAP → G3P (by Triose phosphate isomerase)
G3P → 1,3-Bisphosphoglycerate (by Glyceraldehyde-3-phosphate dehydrogenase)
1,3-Bisphosphoglycerate → 3-Phosphoglycerate (by Phosphoglycerate kinase)
3-Phosphoglycerate → 2-Phosphoglycerate (by Phosphoglycerate mutase)
2-Phosphoglycerate → Phosphoenolpyruvate (PEP) (by Enolase)
Phosphoenolpyruvate (PEP) → Pyruvate (by Pyruvate kinase)
4. Irreversible steps in glycolysis – which ones and why are they irreversible?
There are three irreversible steps in glycolysis, and these steps have high energy changes, making them difficult to reverse. These are:
Hexokinase (Glucose → Glucose-6-phosphate)
Phosphofructokinase-1 (PFK-1) (Fructose-6-phosphate → Fructose-1,6-bisphosphate)
Pyruvate kinase (Phosphoenolpyruvate → Pyruvate)
They are irreversible because these reactions involve a significant release of energy, making it energetically unfavorable to reverse them. These steps essentially "lock" glycolysis in one direction.
5. Steps that consume ATP, steps that produce ATP:
Consume ATP:
Hexokinase step: Glucose → Glucose-6-phosphate (1 ATP consumed)
Phosphofructokinase-1 (PFK-1) step: Fructose-6-phosphate → Fructose-1,6-bisphosphate (1 ATP consumed)
Produce ATP:
Phosphoglycerate kinase step: 1,3-Bisphosphoglycerate → 3-Phosphoglycerate (1 ATP produced)
Pyruvate kinase step: Phosphoenolpyruvate → Pyruvate (1 ATP produced)
Net ATP production in glycolysis is 2 ATP (since 2 ATP are used and 4 ATP are produced).
6. How is Phosphofructokinase-1 (PFK-1) regulated?
PFK-1 is the main regulatory enzyme in glycolysis and is controlled by the energy status of the cell. It is regulated by several factors:
Activated by:
AMP (indicating low energy): If the cell needs more energy, AMP tells PFK-1 to speed up glycolysis.
Fructose-2,6-bisphosphate: This molecule also activates PFK-1, promoting glycolysis.
Inhibited by:
ATP (high energy): If there is plenty of ATP, glycolysis doesn't need to proceed as fast, so ATP inhibits PFK-1.
Citrate (a product of the citric acid cycle): High levels of citrate suggest that energy is plentiful, so PFK-1 is inhibited.
7. What is the glycolysis net reaction?
The net reaction of glycolysis (starting with one molecule of glucose) is: 2 ATP
8. What are the products of anaerobic fermentation?
In anaerobic fermentation (when oxygen is not available), pyruvate produced by glycolysis is converted to different products depending on the organism:
In animals: Pyruvate is converted into lactate (lactic acid) via the enzyme lactate dehydrogenase.
In yeast and some bacteria: Pyruvate is converted into ethanol and carbon dioxide (CO₂) through a two-step process involving pyruvate decarboxylase and alcohol dehydrogenase.
These fermentation pathways regenerate NAD⁺ from NADH, allowing glycolysis to continue in the absence of oxygen.
9. Where are other sugars introduced into glycolysis?
Other sugars, besides glucose, can enter glycolysis at different points:
Fructose: In the liver, fructose is converted into fructose-1-phosphate, which is then broken down into glyceraldehyde and dihydroxyacetone phosphate (DHAP), intermediates in glycolysis.
Galactose: Galactose is converted into glucose-6-phosphate through a series of reactions, allowing it to enter glycolysis at the same point as glucose-6-phosphate.
Mannose: Mannose is converted into fructose-6-phosphate, an intermediate in glycolysis.
These sugars are converted into glycolysis intermediates to be used for energy production.
10. What is the cause of lactose intolerance?
Lactose intolerance occurs when the body lacks sufficient lactase, the enzyme that breaks down lactose (the sugar in milk) into glucose and galactose.
Without enough lactase, lactose cannot be properly digested and absorbed in the small intestine. Instead, it ferments in the colon, causing symptoms like bloating, diarrhea, and gas.
This is a common condition, especially in people of East Asian, African, and Native American descent.
11. Compare glycolysis and gluconeogenesis: energy produced or required.
Glycolysis is an energy-producing pathway:
Net energy: Produces 2 ATP and 2 NADH per glucose molecule.
It breaks down glucose into pyruvate to generate energy.
Gluconeogenesis is the reverse process, used to synthesize glucose:
Energy required: 6 ATP and 2 GTP are used per glucose molecule.
It is an energy-consuming process, as it essentially reverses glycolysis to create glucose from non-carbohydrate precursors (like lactate, amino acids, and glycerol).
Thus, glycolysis generates energy, while gluconeogenesis consumes energy.
12. Complete reversal of glycolysis is not energetically favored; which glycolysis steps block gluconeogenesis and how are these overcome?
The following irreversible steps in glycolysis block its complete reversal into gluconeogenesis:
Hexokinase/Glucokinase: Glucose → Glucose-6-phosphate.
Phosphofructokinase-1 (PFK-1): Fructose-6-phosphate → Fructose-1,6-bisphosphate.
Pyruvate kinase: Phosphoenolpyruvate → Pyruvate.
These blocks are overcome by the following alternate enzymes in gluconeogenesis:
Hexokinase/Glucokinase is bypassed by glucose-6-phosphatase, which removes the phosphate from glucose-6-phosphate to form free glucose.
PFK-1 is bypassed by fructose-1,6-bisphosphatase, which dephosphorylates fructose-1,6-bisphosphate to form fructose-6-phosphate.
Pyruvate kinase is bypassed by a two-step process involving pyruvate carboxylase (which converts pyruvate to oxaloacetate) and PEP carboxykinase (which converts oxaloacetate to phosphoenolpyruvate).
These alternative pathways allow gluconeogenesis to proceed without the irreversible steps of glycolysis.
13. What enzymes and intermediates are different in gluconeogenesis compared to glycolysis?
In gluconeogenesis, the following enzymes are different from those in glycolysis:
Pyruvate → Phosphoenolpyruvate:
Pyruvate carboxylase and PEP carboxykinase bypass the pyruvate kinase step in glycolysis.
Fructose-1,6-bisphosphate → Fructose-6-phosphate:
Fructose-1,6-bisphosphatase bypasses PFK-1 in glycolysis.
Glucose-6-phosphate → Glucose:
Glucose-6-phosphatase bypasses hexokinase in glycolysis.
These enzymes help gluconeogenesis bypass the irreversible steps of glycolysis, allowing for glucose synthesis.
14. What molecules feed into the gluconeogenesis path and what is their source?
Several molecules can be used to generate glucose through gluconeogenesis:
Lactate: Produced during anaerobic fermentation in muscles, lactate can be converted into pyruvate for gluconeogenesis.
Glycerol: Produced from the breakdown of triglycerides (fats), glycerol is converted into dihydroxyacetone phosphate (DHAP), an intermediate in gluconeogenesis.
Amino acids: Specifically, glucogenic amino acids like alanine can be converted into pyruvate or oxaloacetate to enter the gluconeogenesis pathway.
These molecules, often derived from muscle or fat stores, are critical in maintaining blood glucose levels during fasting or low-carbohydrate states.
15. How do glucose and energy charge (ATP) regulate the balance between glycolysis and gluconeogenesis?
The balance between glycolysis and gluconeogenesis is tightly regulated based on the cell’s energy needs and glucose availability:
High ATP and high glucose levels promote gluconeogenesis, since the cell does not need to break down glucose for energy.
Low ATP or low glucose levels favor glycolysis, as the cell needs to generate ATP by breaking down glucose.
Regulatory mechanisms include:
Insulin promotes glycolysis and inhibits gluconeogenesis when glucose is abundant.
Glucagon stimulates gluconeogenesis during fasting or low glucose levels.
AMP (indicating low energy) activates glycolysis and inhibits gluconeogenesis.
Citrate (an intermediate in the citric acid cycle) and ATP inhibit glycolysis and stimulate gluconeogenesis when energy is sufficient.
These regulatory mechanisms help ensure the cell uses glucose efficiently based on its energy status.
16. What is the role of 2,6-fructose-bisphosphate?
2,6-fructose-bisphosphate plays a key role in regulating glycolysis and gluconeogenesis. It acts as a powerful activator of phosphofructokinase-1 (PFK-1) in glycolysis, which helps break down glucose into energy. It also inhibits fructose-1,6-bisphosphatase in gluconeogenesis, preventing the formation of glucose when it’s not needed. Essentially, 2,6-fructose-bisphosphate makes sure that glycolysis happens when glucose is available and prevents gluconeogenesis in the same situation.
17. Which steps of gluconeogenesis occur in the cytoplasm or the mitochondria?
Gluconeogenesis has steps that occur in both the cytoplasm and mitochondria:
Mitochondria:
The conversion of pyruvate to oxaloacetate by pyruvate carboxylase.
Oxaloacetate is then converted into phosphoenolpyruvate (PEP) by PEP carboxykinase, but this takes place in the cytoplasm after being transported from the mitochondria.
Cytoplasm:
The rest of gluconeogenesis (from PEP to glucose) happens in the cytoplasm, including the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate and glucose-6-phosphate to glucose.
18. What is glycogen?
Glycogen is a storage form of glucose in the body. It is made up of long chains of glucose molecules and is primarily stored in the liver and muscles. When the body needs energy, glycogen is broken down into glucose and released into the bloodstream, or used locally by muscle cells during exercise.
19. What are the products of glycogen degradation?
The breakdown of glycogen (called glycogenolysis) produces glucose-1-phosphate as the primary product. This can then be converted into glucose-6-phosphate, which enters glycolysis for energy production. In the liver, glucose-6-phosphate can be converted into free glucose, which is released into the bloodstream to maintain blood sugar levels.
20. How is phosphorylase activity modulated by a kinase?
Phosphorylase is the enzyme that breaks down glycogen. Its activity is controlled by a process called phosphorylation:
A kinase called protein kinase A (PKA) adds a phosphate group to phosphorylase, activating it to break down glycogen.
This process is often triggered by hormones like glucagon (in the liver) or epinephrine (in muscle), which signal the body to release glucose when needed for energy.
21. What are the phosphorylase regulators in the muscle and in the liver?
The regulation of phosphorylase in muscles and liver is slightly different:
In muscles: AMP (indicating low energy) activates phosphorylase to release glucose for energy during exercise. ATP and glucose-6-phosphate inhibit the enzyme when energy levels are sufficient.
In the liver: Glucagon (a hormone released when blood glucose is low) activates phosphorylase to break down glycogen and release glucose into the blood. Insulin, on the other hand, inhibits glycogen breakdown.
22. How is glycogen synthesis stimulated; how is degradation blocked in the liver during synthesis?
Glycogen synthesis is stimulated by:
Insulin, which is released after meals when glucose levels are high. Insulin promotes the conversion of glucose into glycogen by activating glycogen synthase.
During glycogen synthesis, glycogen degradation is blocked by insulin as well. Insulin inhibits the enzyme glycogen phosphorylase, preventing the breakdown of glycogen.
In the liver, this ensures that glucose is stored as glycogen rather than released into the blood.
23. What roles do glucagon and insulin play in glycogen synthesis?
Insulin: Promotes glycogen synthesis after meals by activating glycogen synthase and inhibiting glycogen phosphorylase (which breaks down glycogen). This helps store glucose as glycogen in the liver and muscles.
Glucagon: Increases glycogen degradation during fasting (when blood glucose is low) by activating glycogen phosphorylase, which breaks down glycogen into glucose for the bloodstream.
In short, insulin stores glucose as glycogen, while glucagon releases glucose from glycogen.
24. What is creatine, how is it made, and what role does it play as an energy source?
Creatine is a compound found in muscles that helps produce energy quickly during short bursts of intense activity:
How it is made: Creatine is synthesized in the liver, kidneys, and pancreas from the amino acids arginine, glycine, and methionine.
Role as an energy source: Creatine is stored in the muscles as creatine phosphate. During intense exercise, creatine phosphate donates its phosphate group to ADP to quickly regenerate ATP, the primary energy carrier in cells. This provides extra energy during activities like sprinting or weightlifting, where energy demand is rapid.
CITRIC ACID CYCLE
1. Where does the Citric Acid Cycle (CAC) occur?
The Citric Acid Cycle (CAC), also called the Krebs Cycle or TCA cycle, occurs in the mitochondria of the cell, specifically in the mitochondrial matrix.
2. What is the step that commits pyruvate to the citric acid cycle?
The step that commits pyruvate to the citric acid cycle is its conversion into acetyl-CoA by the pyruvate dehydrogenase complex (PDC). Once pyruvate is converted to acetyl-CoA, it is ready to enter the citric acid cycle.
3. What are some roles of acetyl CoA?
Acetyl-CoA has several important roles:
It enters the citric acid cycle to generate energy (ATP).
It is used to synthesize fatty acids and cholesterol.
It can be converted into ketone bodies in the liver during fasting or exercise.
It helps in protein synthesis and other biosynthetic processes.
4. How are thiamine, lipoic acid, FAD, and NAD+ used? What vitamins are these?
Thiamine (Vitamin B1): Thiamine is part of thiamine pyrophosphate (TPP), which is used in the pyruvate dehydrogenase complex to convert pyruvate into acetyl-CoA.
Lipoic acid: Lipoic acid is a coenzyme that aids in the pyruvate dehydrogenase complex, helping to transfer electrons and acyl groups.
FAD (Flavin adenine dinucleotide): FAD is a coenzyme used in the citric acid cycle to accept electrons, such as in the reaction catalyzed by succinate dehydrogenase.
NAD+ (Nicotinamide adenine dinucleotide): NAD+ is a coenzyme that accepts electrons in the citric acid cycle, such as in reactions catalyzed by isocitrate dehydrogenase and malate dehydrogenase.
5. How is lipoic acid regenerated in the enzyme?
Lipoic acid is regenerated in the pyruvate dehydrogenase complex after it has been oxidized by accepting electrons. The enzyme dihydrolipoamide dehydrogenase helps regenerate oxidized lipoic acid by transferring electrons from lipoamide to NAD+, converting it back to its active form.
6. In the formation of acetyl CoA from pyruvate, what other products are formed?
When pyruvate is converted into acetyl-CoA by the pyruvate dehydrogenase complex, NAD+ is reduced to NADH and CO2 is released as a byproduct. Therefore, the products of the reaction are acetyl-CoA, NADH, and CO2.
7. Describe the pyruvate dehydrogenase complex and its regulation.
The pyruvate dehydrogenase complex (PDC) is a large enzyme complex that converts pyruvate into acetyl-CoA, which then enters the citric acid cycle. The complex consists of multiple subunits and requires several cofactors like thiamine pyrophosphate (TPP), lipoic acid, FAD, and NAD+. The activity of PDC is tightly regulated:
Phosphorylation by pyruvate dehydrogenase kinase (PDK) inhibits the complex.
Dephosphorylation by pyruvate dehydrogenase phosphatase activates the complex.
This regulation ensures that the complex only works when the cell needs energy.
8. What does thiamine deficiency cause and how can this be prevented?
A thiamine deficiency can lead to diseases like beriberi and Wernicke-Korsakoff syndrome, both of which involve neurological and cardiovascular problems. Thiamine is essential for the pyruvate dehydrogenase complex and other enzymes, so deficiency can disrupt energy production. It can be prevented by ensuring an adequate intake of thiamine through food (like whole grains, pork, and legumes) or supplements.
9. How is the CAC coupled to oxygen consumption?
The citric acid cycle (CAC) is indirectly coupled to oxygen consumption because the NADH and FADH2 produced during the cycle carry electrons to the electron transport chain (ETC), where oxygen is the final electron acceptor. Oxygen is needed to drive the electron flow through the ETC, which generates a proton gradient used to produce ATP. Without oxygen, the cycle cannot run efficiently, as the NADH and FADH2 would accumulate.
10. Be able to recognize the structures, names, and enzymes of the citric acid cycle.
The citric acid cycle involves a series of reactions with the following key molecules and enzymes:
Acetyl-CoA + oxaloacetate → citrate (enzyme: citrate synthase)
Citrate → isocitrate (enzyme: aconitase)
Isocitrate → alpha-ketoglutarate (enzyme: isocitrate dehydrogenase)
Alpha-ketoglutarate → succinyl-CoA (enzyme: alpha-ketoglutarate dehydrogenase)
Succinyl-CoA → succinate (enzyme: succinyl-CoA synthetase)
Succinate → fumarate (enzyme: succinate dehydrogenase)
Fumarate → malate (enzyme: fumarase)
Malate → oxaloacetate (enzyme: malate dehydrogenase)
11. Which steps are irreversible (drive the pathway); which steps are regulated directly and how?
The irreversible steps in the citric acid cycle are:
Citrate synthase (acetyl-CoA + oxaloacetate → citrate)
Isocitrate dehydrogenase (isocitrate → alpha-ketoglutarate)
Alpha-ketoglutarate dehydrogenase (alpha-ketoglutarate → succinyl-CoA)
These steps are regulated directly by feedback inhibition:
ATP and NADH inhibit isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase when energy levels are high.
ADP and Ca2+ activate these enzymes when the cell needs more energy.
12. What is the net reaction of the citric acid cycle?
The net reaction of the citric acid cycle per acetyl-CoA molecule is:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → 2 CO2 + 3 NADH + FADH2 + GTP + CoA
This reaction produces 3 NADH, 1 FADH2, 1 GTP (or ATP), and 2 CO2 molecules per turn of the cycle.
13. Indirect regulation can occur when intermediates are siphoned off for other paths; which intermediates are removed to make other biomolecules?
Certain intermediates of the citric acid cycle can be removed for other pathways:
Citrate can be used to make fatty acids and cholesterol.
Alpha-ketoglutarate is used in the synthesis of amino acids like glutamate.
Succinyl-CoA can be used for heme production.
Oxaloacetate can be used to make glucose via gluconeogenesis.
These intermediates are siphoned off when the body needs them for biosynthesis, and this can indirectly regulate the citric acid cycle.
Oxidative Phosphorylation
1. What is the structure of the mitochondria?
The mitochondria are often described as "bean-shaped" or "football-shaped" organelles within cells. They are surrounded by two membranes:
Outer membrane: Smooth and semi-permeable.
Inner membrane: Highly folded into structures called cristae, which increase the surface area. The space inside the inner membrane is called the matrix. The inner membrane is where most of the important reactions for energy production (like the electron transport chain and ATP synthesis) take place.
2. What governs the flow of electrons?
The flow of electrons is governed by the electron transport chain (ETC). Electrons are passed through a series of protein complexes and carriers, starting with NADH and FADH2, and eventually transferring to oxygen (O2), which is the final electron acceptor. This flow of electrons generates a proton gradient (protons pumped across the inner mitochondrial membrane).
3. What and where are the carriers that transfer electrons from NADH/FADH2 to O2?
The main carriers that transfer electrons from NADH and FADH2 to oxygen (O2) are proteins in the inner mitochondrial membrane, part of the electron transport chain:
Complex I (NADH dehydrogenase): Receives electrons from NADH.
Complex II (Succinate dehydrogenase): Receives electrons from FADH2.
Coenzyme Q (CoQ or ubiquinone): Transfers electrons from complexes I and II to Complex III.
Cytochrome C: Transfers electrons from Complex III to Complex IV.
Complex IV (Cytochrome C oxidase): Transfers electrons to oxygen, forming water (H2O).
4. Which electron transfers result in protons being pumped across the inner membrane?
Protons (H+) are pumped across the inner mitochondrial membrane by the following complexes:
Complex I (NADH dehydrogenase): Pumps protons across the membrane as it transfers electrons from NADH to CoQ.
Complex III (Cytochrome bc1 complex): Pumps protons across the membrane as it transfers electrons from CoQ to cytochrome C.
Complex IV (Cytochrome C oxidase): Also pumps protons while transferring electrons to oxygen. These proton pumps create an electrochemical gradient known as the proton motive force.
5. Why is it necessary for the proteins involved in harvesting electrons to be membrane proteins?
The proteins involved in electron transport need to be membrane-bound because the electron transport chain happens in the inner mitochondrial membrane. This allows the proteins to pass electrons through the membrane and simultaneously pump protons into the intermembrane space, creating the proton gradient needed to produce ATP.
6. What is the proton motive force?
The proton motive force (PMF) is the energy stored in the form of a proton gradient across the inner mitochondrial membrane. This gradient is created by the electron transport chain, which pumps protons from the matrix into the intermembrane space. The PMF drives the enzyme ATP synthase, which uses the flow of protons back into the matrix to produce ATP.
7. What is the ATPase; how do the domains work together?
The ATPase (specifically ATP synthase) is an enzyme that produces ATP from ADP and phosphate (Pi). It has two main parts:
F0 domain: The part that spans the membrane and forms a channel for protons to flow through.
F1 domain: The part that sticks into the matrix and produces ATP by using the energy from proton flow through the F0 domain.
As protons flow through the F0 domain, it causes the F1 domain to rotate, which helps convert ADP and Pi into ATP.
8. How does the ATPase motor work?
The ATPase motor works by using the proton motive force (PMF). As protons flow through the F0 domain of ATP synthase, it causes the F1 domain to rotate. This rotational movement helps bind ADP and Pi together to form ATP. The flow of protons through ATP synthase is essentially the "motor" that powers ATP production.
9. How many ATP are produced by NADH and FADH2?
Each NADH molecule produces approximately 2.5 ATP.
Each FADH2 molecule produces approximately 1.5 ATP. These ATP are produced through the electron transport chain and oxidative phosphorylation, using the energy from NADH and FADH2 to pump protons and generate the proton motive force, which drives ATP synthase.
10. How does the level of ADP control O2 consumption?
The level of ADP directly controls the rate of oxygen consumption. When ADP levels are high, it signals that the cell needs more ATP. This activates ATP synthase, increasing the flow of protons through the enzyme and speeding up the electron transport chain, which in turn increases oxygen consumption (since oxygen is the final electron acceptor). When ADP levels are low, oxygen consumption decreases as less ATP is needed.
11. Names of the activated carriers: FAD, NAD+, acetyl CoA; what job does each do and where does each one come from (vitamin)?
FAD (Flavin adenine dinucleotide): Carries electrons in the form of FADH2 in reactions like the citric acid cycle. It is derived from Vitamin B2 (riboflavin).
NAD+ (Nicotinamide adenine dinucleotide): Carries electrons in the form of NADH in processes like glycolysis, the citric acid cycle, and the electron transport chain. It is derived from Vitamin B3 (niacin).
Acetyl-CoA: Delivers two-carbon units to the citric acid cycle for energy production. It is derived from the breakdown of glucose, fatty acids, and amino acids.
12. What are all of the molecules that can carry electrons?
The main molecules that can carry electrons in cellular processes are:
NADH (from NAD+)
FADH2 (from FAD)
Coenzyme Q (Ubiquinone): A small molecule that transfers electrons in the electron transport chain.
Cytochrome C: A protein that transfers electrons between Complex III and Complex IV in the electron transport chain.
Iron-sulfur clusters: Found in certain enzymes and electron transport chain complexes, these clusters help transfer electrons.
Copper ions: Present in Complex IV of the electron transport chain, copper also helps in electron transfer.
Lipids
1. Where are fatty acids (FA) stored as an energy reserve?
Fatty acids are stored as triacylglycerols (TAG) in adipose tissue (fat cells) in the body. These triglycerides are a major form of energy storage.
2. What are the products when triacylglycerol is degraded and what is the fate of glycerol?
When triacylglycerol (TAG) is broken down (a process called lipolysis), it produces:
Glycerol, which can be converted into glucose through gluconeogenesis in the liver.
Fatty acids, which are transported to the mitochondria for energy production.
3. What are the three processes for complete oxidation of fatty acid to CO2 and H2O (producing energy)?
The complete oxidation of fatty acids happens in three main processes:
Beta-oxidation: Fatty acids are broken down into acetyl-CoA molecules in the mitochondria.
Citric Acid Cycle (CAC): Acetyl-CoA enters the citric acid cycle to generate high-energy molecules (NADH, FADH2, and GTP).
Oxidative phosphorylation: NADH and FADH2 produced in the citric acid cycle pass electrons through the electron transport chain to generate ATP, with oxygen as the final electron acceptor (producing water).
4. Why is fatty acid degradation referred to as beta-oxidation?
Fatty acid degradation is called beta-oxidation because the process involves the removal of two-carbon units from the fatty acid at the beta carbon (the second carbon in the fatty acid chain). This results in the production of acetyl-CoA.
5. What drives the reaction of acyl-CoA synthetase?
The enzyme acyl-CoA synthetase drives the activation of fatty acids by attaching CoA (coenzyme A) to the fatty acid. This reaction is powered by the energy released from the hydrolysis of ATP into AMP and PPi (pyrophosphate), essentially "activating" the fatty acid for degradation.
6. What is the function of carnitine?
Carnitine is a molecule that helps transport fatty acids into the mitochondria. It binds to acyl-CoA (fatty acids attached to coenzyme A) and allows it to pass through the mitochondrial membrane, where it can then undergo beta-oxidation for energy production.
7. Be able to identify the four recurring steps of FA degradation (oxidation, hydration, oxidation, thiolysis) and know that there are two carbons removed in each round.
The four recurring steps of fatty acid degradation (beta-oxidation) are:
Oxidation: The fatty acid (acyl-CoA) is oxidized by FAD, producing FADH2.
Hydration: Water is added to the molecule to form a hydroxyacyl-CoA.
Oxidation: The hydroxy group is oxidized by NAD+, producing NADH.
Thiolysis: The bond between carbons is cleaved by CoA, releasing a two-carbon unit in the form of acetyl-CoA. This is the key step, and two carbons are removed in each round.
8. What is the net reaction of fatty acid degradation?
The net reaction of fatty acid degradation (for one round of beta-oxidation) is:
Acyl-CoA → Acetyl-CoA + FADH2 + NADH. For a fatty acid with an even number of carbon atoms, the process repeats until the entire chain is broken down into acetyl-CoA units.
9. How does citrate provide both electrons (NADH and NADPH) and carbon (acetyl-CoA) for fatty acid synthesis?
Citrate is a key molecule that connects the citric acid cycle with fatty acid synthesis:
Citrate leaves the mitochondria and enters the cytoplasm.
In the cytoplasm, citrate is converted into acetyl-CoA (which provides the two-carbon units for fatty acid synthesis).
Acetyl-CoA can then be used to generate NADPH, a reducing agent required for fatty acid synthesis, through reactions involving the enzyme ATP-citrate lyase.
10. How is CO2 used in fatty acid synthesis?
In fatty acid synthesis, CO2 is used to convert acetyl-CoA into a molecule called malonyl-CoA. This is the first step of fatty acid synthesis and is catalyzed by the enzyme acetyl-CoA carboxylase. The addition of CO2 to acetyl-CoA is an important regulatory step.
11. What cofactor does acyl carrier protein require?
The acyl carrier protein (ACP) requires a phosphopantetheine group, which is derived from vitamin B5 (pantothenic acid). This cofactor helps ACP hold onto the growing fatty acid chain during synthesis, facilitating the process.
12. Be able to identify the four recurring steps of FA synthesis [condensation (synthesis), reduction, dehydration, reduction].
The four recurring steps of fatty acid synthesis are:
Condensation (Synthesis): Acetyl-CoA and malonyl-CoA are joined together to form a 4-carbon chain.
Reduction: The newly formed carbon chain is reduced by NADPH to form a saturated intermediate.
Dehydration: Water is removed to form a double bond in the fatty acid chain.
Reduction: The double bond is reduced again using NADPH, completing the fatty acid chain.
13. Know that all of the enzymes for fatty acid synthesis are domains in one long polypeptide chain.
All the enzymes involved in fatty acid synthesis are part of a single polypeptide chain called fatty acid synthase. This large enzyme complex has multiple domains, each responsible for a different step in the synthesis of fatty acids.
14. Aspirin, ibuprofen, and acetaminophen block what enzyme by mimicking the product prostaglandin?
Aspirin, ibuprofen, and acetaminophen block the enzyme cyclooxygenase (COX). COX is involved in the production of prostaglandins, which are molecules that promote inflammation and pain. These drugs work by mimicking prostaglandins, effectively inhibiting the enzyme and reducing inflammation.
15. What regulates fatty acid synthesis?
Fatty acid synthesis is regulated by several factors:
Acetyl-CoA carboxylase (ACC): The enzyme that catalyzes the conversion of acetyl-CoA to malonyl-CoA. It is activated by citrate and insulin (when energy is abundant) and inhibited by palmitoyl-CoA (the end product of fatty acid synthesis).
Insulin promotes fatty acid synthesis when energy is plentiful.
Glucagon and epinephrine inhibit fatty acid synthesis when energy is needed.
Determination of Macromolecular Structure
1. What are ways to determine the 3D structure of macromolecules?
There are several ways to determine the 3D structure of macromolecules (like proteins, DNA, and RNA). Some of the main methods include:
X-ray Crystallography: A technique where a molecule is crystallized, and then X-rays are passed through the crystals to create a diffraction pattern. This pattern is used to determine the 3D arrangement of atoms.
Cryo-Electron Microscopy (Cryo-EM): The sample is frozen and then examined under an electron microscope. It provides high-resolution images of the 3D shape of large molecules, especially those that can't be crystallized.
Nuclear Magnetic Resonance (NMR): A technique that uses magnetic fields and radio waves to determine the structure of molecules in solution (not in a crystal).
Electron Microscopy (EM): A method that uses electrons instead of light to create detailed images of macromolecules, often at lower resolutions than X-ray crystallography or Cryo-EM.
Computational Modeling: Using computer simulations to predict the 3D structure of molecules based on known data or evolutionary relationships.
2. How does resolution and contrast impact macromolecular structure determination?
Resolution refers to how clear and detailed the images are. High resolution gives a more accurate, precise view of the structure, allowing you to see small details like side chains of amino acids or fine atomic positions. Low resolution results in a blurry or less precise model.
Contrast refers to the difference in intensity between different parts of an image. High contrast helps highlight features of the macromolecule (like a protein's core vs. its surface), making it easier to distinguish details. Low contrast might make the structure harder to visualize or interpret.
Both resolution and contrast are critical for obtaining clear, accurate, and useful 3D structures.
3. Describe how protein models are generated and where 3D structures may be visualized.
Protein models are generated by combining experimental data (like X-ray diffraction or NMR) with computational techniques. Here's how:
Experimental data: Techniques like X-ray crystallography or Cryo-EM give information about the positions of atoms in the protein.
Computational modeling: This data is used to generate a model that best fits the experimental results.
Refinement: The model is adjusted and refined until it matches the experimental data as closely as possible.
3D protein structures can be visualized using software tools like:
PyMOL
Chimera
RasMol These programs allow researchers to view and manipulate the 3D models of proteins to study their structure and function.
4. How has AlphaFold helped the field of structural biology?
AlphaFold is an AI-based program developed by DeepMind that predicts the 3D structure of proteins from their amino acid sequences. It has significantly advanced the field by:
Providing highly accurate predictions of protein structures where experimental data (like X-ray or Cryo-EM) is difficult or time-consuming to obtain.
Helping scientists understand protein folding, which is crucial for many biological processes and diseases (like Alzheimer's).
AlphaFold has been a breakthrough in structural biology because it can predict protein structures in a way that was previously not possible with high accuracy.
5. Describe the rationale and process of negative stain electron microscopy. How is this different from Cryo-electron microscopy?
Negative Stain Electron Microscopy: This method involves applying a heavy metal stain (like uranyl acetate) to the sample. The stain does not bind to the macromolecule but surrounds it, making the molecule appear lighter than the background in the electron microscope. This technique is used to get detailed images of larger complexes but usually at lower resolution.
Rationale: The stain highlights the boundaries of the macromolecule, making it stand out against the background.
Difference from Cryo-EM: In negative stain EM, the sample is stained and dried, so it may lose some of its natural state. In Cryo-EM, the sample is frozen without staining, preserving its natural structure in a near-native state, and often gives better resolution.
6. How is an X-ray diffraction experiment similar to a Cryo-EM experiment and how are they different?
Similarities:
Both X-ray diffraction and Cryo-EM are used to determine the 3D structure of macromolecules.
Both methods involve collecting data from the sample and using that data to reconstruct the molecule's structure.
Differences:
X-ray diffraction requires the macromolecule to be crystallized. This can be challenging because not all molecules can form high-quality crystals.
Cryo-EM, on the other hand, involves flash-freezing the sample and analyzing it directly in its natural, non-crystalline state, making it easier for larger, more complex molecules that don't easily form crystals.
Resolution: X-ray crystallography typically provides higher resolution data than Cryo-EM, but Cryo-EM has improved significantly in recent years and can now achieve very high-resolution structures.
Amino Acids and Nucleic Acids
1. In the breakdown of amino acids, all amino acids transfer ammonia to glutamate.
During the breakdown of amino acids, their amino group (NH2) is removed and transferred to glutamate. This process is called transamination. Glutamate then carries the ammonia (NH3), which can be safely transported to the liver for processing.
2. What are the products of alanine aminotransferase?
Alanine aminotransferase (ALT) is an enzyme that helps transfer an amino group from alanine to alpha-ketoglutarate. The products of this reaction are:
Glutamate (which gets the amino group).
Pyruvate (which comes from alanine after losing the amino group).
3. What is the net reaction of amino acid to ammonia?
The net reaction of amino acid breakdown to ammonia involves the removal of the amino group from the amino acid. Here's a simplified view:
Amino acid + Alpha-ketoglutarate → Glutamate + Alpha-keto acid.
Then, glutamate can be converted into ammonia (NH3), which is released in the urea cycle in the liver.
4. Why is glutamate converted to glutamine as a nitrogen carrier?
Glutamate is converted to glutamine by adding an extra ammonia (NH3) molecule. This conversion helps safely carry ammonia in the blood to the liver. Glutamine is more stable and non-toxic compared to free ammonia, which can be harmful to the body.
5. The urea cycle occurs in the liver.
The urea cycle (also known as the ornithine cycle) happens in the liver. It is a process where ammonia (toxic in high amounts) is converted into urea, which is then excreted from the body through the urine.
6. How is the urea cycle linked to glycolysis/gluconeogenesis and the citric acid cycle (CAC)?
The urea cycle is connected to glycolysis/gluconeogenesis and the citric acid cycle (CAC) in the following ways:
Glycolysis produces pyruvate, which can be converted into acetyl-CoA, entering the citric acid cycle (CAC).
The CAC produces oxaloacetate, which is a key intermediate for the urea cycle.
Some of the amino acids used in the urea cycle are derived from intermediates in glycolysis and the citric acid cycle, linking energy production with nitrogen disposal.
7. What do gluconeogenic and ketogenic mean?
Gluconeogenic refers to amino acids that can be converted into glucose (a sugar), which the body can use for energy. These amino acids are often used in gluconeogenesis, the process of making glucose from non-carbohydrate sources.
Ketogenic refers to amino acids that can be converted into ketone bodies, which are used as an alternative energy source, especially during fasting or low-carb diets. These amino acids are often converted into acetyl-CoA.
8. When carbon is harvested from amino acids, what products are formed?
When the carbon from amino acids is used for energy, the products formed depend on the type of amino acid:
Gluconeogenic amino acids: These are converted into glucose (or intermediates for glucose production).
Ketogenic amino acids: These are converted into acetyl-CoA, which can be used to make ketone bodies.
Some amino acids can also feed into the citric acid cycle (CAC), contributing to ATP production.
KEY CONCEPTS:
1. Triacylglyceride Digestion in Small Intestine
Triacylglycerides (TAGs) are broken down into:
Monoglycerides and Fatty acids by the enzyme pancreatic lipase.
These breakdown products are absorbed into the intestinal cells, where they are re-esterified to form chylomicrons and transported to the lymphatic system.
2. Muscle Fiber Contraction and Glycolysis
During muscle fiber contraction, the demand for ATP increases.
Pyruvate kinase activity is increased to facilitate the conversion of phosphoenolpyruvate (PEP) to pyruvate, generating ATP in the process.
Glycolysis is stimulated because it provides quick ATP production by breaking down glucose to pyruvate, which is crucial for sustained muscle contraction during exercise.
3. Urea Synthesis and Glucose Levels
Urea synthesis occurs in the liver via the urea cycle, utilizing aspartate and ATP.
Increased urea production requires more ATP, which may lead to increased glucose production through gluconeogenesis to supply the necessary ATP for the urea cycle, raising glucose levels in cells.
4. Anaerobic Glycolysis
Under anaerobic conditions, NAD+ is regenerated through the conversion of pyruvate to lactate via lactate dehydrogenase. This step allows glycolysis to continue, producing ATP in the absence of oxygen. This process is crucial in tissues like muscle during intense exercise, when oxygen cannot be supplied fast enough.
5. α-Ketoglutarate Structure
α-Ketoglutarate is a key intermediate in the citric acid cycle (TCA cycle). Its structure includes:
A 5-carbon chain with a carboxyl group at the end and a ketone group at the second carbon. It’s important to understand this structure as it plays a role in both the TCA cycle and amino acid metabolism.
6. Why the Citric Acid Cycle Requires Aerobic Conditions
The citric acid cycle requires oxygen indirectly because it depends on the electron transport chain (ETC) to regenerate NAD+ and FAD from NADH and FADH2, respectively. These coenzymes are used in the TCA cycle to facilitate the oxidation of intermediates. Without oxygen, the ETC cannot function, leading to a buildup of NADH and FADH2, halting the TCA cycle.
7. Glycolysis Enzyme Functions
Enzymes involved in glycolysis and their functions:
Hexokinase: Converts glucose to glucose-6-phosphate, trapping glucose inside the cell.
Phosphofructokinase-1 (PFK-1): Catalyzes the rate-limiting step, converting fructose-6-phosphate to fructose-1,6-bisphosphate.
Aldolase: Cleaves fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Glyceraldehyde 3-phosphate dehydrogenase: Converts glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate while reducing NAD+ to NADH.
Phosphoglycerate kinase: Generates ATP by converting 1,3-bisphosphoglycerate to 3-phosphoglycerate.
Pyruvate kinase: Converts PEP to pyruvate, generating ATP in the process.
8. Ketogenic Amino Acid Degradation
Ketogenic amino acids (e.g., leucine, lysine) contribute carbon to form acetyl-CoA and acetoacetate, intermediates in ketone body formation. These products are important for energy production during fasting or prolonged exercise.
9. Irwin Rose and the Proteasome
Irwin Rose’s discovery of ubiquitin and the proteasome system plays a critical role in protein degradation.
The proteasome degrades proteins that are tagged with ubiquitin. This system helps regulate protein levels in the cell, remove misfolded proteins, and control cell cycle progression.
10. Beta-Oxidation of Fatty Acids
Beta-oxidation refers to the oxidative degradation of fatty acids. The process involves the β-carbon (the carbon two positions away from the carboxyl group) being oxidized in multiple steps to form acetyl-CoA, which can enter the citric acid cycle for further energy production.
11. Pyruvate Dehydrogenase Complex
The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate into acetyl-CoA, committing the carbon atoms to either oxidation in the citric acid cycle or fatty acid synthesis.
Stimulated by: AMP, CoA (increased energy demand).
Inhibited by: NADH, acetyl-CoA (feedback inhibition indicating sufficient energy supply).
12. Fatty Acid Synthesis and HCO3-
HCO3- (bicarbonate) is required in fatty acid synthesis to convert acetyl-CoA into malonyl-CoA using the enzyme acetyl-CoA carboxylase. However, the carbon from bicarbonate does not appear in the final fatty acid product because it is used in the malonyl-CoA intermediate and then lost during the elongation cycle.
13. Glycogen and Glycogen Degradation
Glycogen is a branched polysaccharide composed of glucose units, primarily stored in the liver and muscle.
Hormones that stimulate glycogen degradation include glucagon (in liver) and epinephrine (in muscle), both activating glycogen phosphorylase.
14. The Matrix and Energy Generation
In “The Matrix”, disrupting mitochondrial membrane proteins (such as by removing electrons directly) would block oxidative phosphorylation, halting ATP production. Mitochondria play a central role in aerobic energy production by using oxygen to generate ATP via the electron transport chain.
15. Insulin and Blood Glucose Levels
Insulin is a hypoglycemic hormone that lowers blood glucose levels by promoting glucose uptake into cells and stimulating glycogen synthesis.
16. Net Reactions for Glycolysis and Gluconeogenesis
Glycolysis: Glucose+2NAD++2ADP+2Pi→2Pyruvate+2NADH+2ATP\text{Glucose} + 2 \text{NAD}^+ + 2 \text{ADP} + 2 \text{P}_i \rightarrow 2 \text{Pyruvate} + 2 \text{NADH} + 2 \text{ATP}Glucose+2NAD++2ADP+2Pi→2Pyruvate+2NADH+2ATP
Gluconeogenesis: 2Pyruvate+4ATP+2GTP+2NADH+6H2O→Glucose+4ADP+2GDP+6Pi+2NAD+2 \text{Pyruvate} + 4 \text{ATP} + 2 \text{GTP} + 2 \text{NADH} + 6 \text{H}_2\text{O} \rightarrow \text{Glucose} + 4 \text{ADP} + 2 \text{GDP} + 6 \text{Pi} + 2 \text{NAD}^+2Pyruvate+4ATP+2GTP+2NADH+6H2O→Glucose+4ADP+2GDP+6Pi+2NAD+
ATP/GTP usage: Glycolysis uses 2 ATP and 2 NADH, while gluconeogenesis requires 4 ATP and 2 GTP for the reverse process.
17. Citric Acid Cycle and Tricarboxylate Cycle
Molecules with three carboxyl groups:
Citrate and isocitrate (both are intermediates in the citric acid cycle).
18. Fatty Acid Synthesis Reaction
Fatty acid synthesis involves acetyl-CoA being converted to malonyl-CoA by acetyl-CoA carboxylase (requiring HCO3- and ATP).
19. ATP Hydrolysis and Energy Release
ATP hydrolysis releases energy for three main reasons:
Electrostatic Repulsion: Negative charges on the phosphate groups repel each other.
Resonance Stabilization: The products (ADP and Pi) are more stable due to resonance.
Increased Hydration: Water molecules interact more favorably with the products.
20. Metabolic Pathways and Enzymes
Glycolysis, gluconeogenesis, and fatty acid metabolism all occur in the cytosol.
The citric acid cycle occurs in the mitochondria.
ATP-producing enzymes in glycolysis include phosphoglycerate kinase and pyruvate kinase.
21. Regulation of Phosphofructokinase-1
Inhibitors of PFK-1:
ATP and citrate (signaling sufficient energy).
Activators of PFK-1:
AMP, fructose 2,6-bisphosphate (signaling low energy).
22. Gluconeogenesis and Phosphoenolpyruvate
Phosphoenolpyruvate (PEP) is generated from oxaloacetate by phosphoenolpyruvate carboxykinase (PEPCK) during gluconeogenesis.
23. Ketone Body Formation and Carnitine
Ketone bodies are produced from acetyl-CoA during fasting or low carbohydrate diets.
Carnitine is essential for the transport of long-chain fatty acids into the mitochondria for β-oxidation.
24. Protein Degradation and Ubiquitination
Ubiquitin tags proteins for degradation in the proteasome, which recognizes and breaks down the proteins, ensuring proper regulation of protein turnover.
25. Anaerobic Fermentation Products
The products of anaerobic fermentation in muscle cells are lactate (lactic acid) and ethanol (in yeast).