BIO 3C EXAM 2

Explain the role of GLUT transporters.

(You don't need to know what each one does, just what a GLUT transporter is). How does glucose get into cells in the first place? Well, this family of proteins sits at the plasma membrane, "Glucose transporters". They mediate the thermodynamically downhill movement of glucose across the plasma membrane. They are located on the plasma membrane of all mammalian tissues. * GLUT 2: glucose enters into these tissues only when [glucose] is high in the blood. * GLUT 4: Insulin will serve as the ligand of a signal transduction pathway that will send more GLUT 4 receptors to the cell surface (promoting uptake of glucose by muscle and fat).

Be familiar with Insulin Signaling

* A second example of an RTK (receptor tyosine kinase) * Via a receptor tyrosine kinase * Insulin signaling will trigger the movement of glucose receptors such as GLUT4, to the cell membrane so that they can take glucose in, which will trigger the store of glucose as glycogen, it'll promote glycogen synthesis. Insulin signaling inhibits degradation of fat and promotes fat synthesis. * Insulin is the ligand, it will bind to the receptor (blue) at the α subunit. There's the transmembrane domain (the part of the yellow within the membrane), and then the intracellular domain (yellow outside the membrane). The yellow is the β subunit. Binding of insulin results in cross-phosphorylation, and then the phosphorylated sites on the insulin receptor act as binding sites for the protein known as the insulin receptor substrate, (or IRS-1). Then, the IRS protein will be phosphorylated, again by the tyosine kinase activity of the insulin receptor. Now that its phosphorylated, it'll act as an adapter protein. These phosphotyrosine residues will be recognized by other proteins including (shown in green) the lipid kinase that will phosphorylate PIP2, to form PIP3. PIP3 activates PDK1 (PIP3-dependent protein kinase 1), which is yet another kinase that can then phosphorylate and activate a protein known as Akt. Akt is a serine/threonine kinase. It'll phosphorylate enzymes that stimulate glycogen synthesis, and it'll also phosphorylate components to control the trafficking of GLUT4 to the cell surface. When we have more GLUT4 receptors at the surface of our cells, we can bring glucose into cells and get glucose out of the blood. Proteins phosphorylated on serine, threonine, or tyrosine residues, such as insulin-receptor substrates, do not hydrolyze spontaneously; they are extremely stable kinetically. Specific enzymes, called protein phosphatases, are required to hydrolyze these phosphorylated proteins and convert them back into the states they were in before the initiation of signaling. Similarly, lipid phosphatases are required to remove phosphoryl groups from inositol lipids that had been phosphorylated as part of a signaling cascade. In insulin signaling, three classes of enzymes are of particular note: (1) protein tyrosine phosphatases that remove phosphoryl groups from tyrosine residues on the insulin receptor, (2) lipid phosphatases that hydrolyze PIP3 to PIP2, and (3) protein serine phosphatases that remove phosphoryl groups from activated protein kinases such as Akt. Many of these phosphatases are activated or recruited as part of the response to insulin. Thus, binding of the initial signal sets the stage for the eventual termination of the response. Insulin receptor: IRS-1 -> PIP3 kinase -> PDK-1 -> Akt

Draw the complete pathway for glycolysis naming all enzymes and substrates. Be able to recognize structures.

* Glycolysis: the sequence of reactions that convert one molecule of glucose into two molecules of pyruvate while generating ATP.

What role do protein kinases and phosphatases play in signal transduction?

* Kinases are enzymes that phosphorylate a substrate at the expense of a molecule of ATP (Serine/Threonine/Tyrosine residues (-OH)). In the insulin pathway, PIP2 is phosphorylated by PI3K (phosphatidylinositide-3-kinase). In this phosphorylated state, PIP2 is known as PIP3. Even though PIP2 isn't a protein, so it doesn't have those ser/thr/tyr residues to phosphorylate, it still has an exposed -OH group, which is what gets phosphorylated on those amino acid residues anyways, which is why PIP2 is able to be phosphorylated. * We have seen that the activated G protein promotes its own inactivation by the release of a phosphoryl group from GTP. In contrast, proteins phosphorylated on serine, threonine, or tyrosine residues, such as insulin-receptor substrates, do not hydrolyze spontaneously; they are extremely stable kinetically. Specific enzymes, called protein phosphatases, are required to hydrolyze these phosphorylated proteins and convert them back into the states they were in before the initiation of signaling. Similarly, lipid phosphatases are required to remove phosphoryl groups from inositol lipids that had been phosphorylated as part of a signaling cascade. In insulin signaling, three classes of enzymes are of particular note: (1) protein tyrosine phosphatases that remove phosphoryl groups from tyrosine residues on the insulin receptor, (2) lipid phosphatases that hydrolyze PIP3 to PIP2, and (3) protein serine phosphatases that remove phosphoryl groups from activated protein kinases such as Akt. Many of these phosphatases are activated or recruited as part of the response to insulin. Thus, binding of the initial signal sets the stage for the eventual termination of the response.

Describe the reciprocal regulation of glycolysis and gluconeogenesisi. Include a description of the role of fructose 2,6-bisphosphate in your answer.

* The basic premise of the reciprocal regulation is that when glucose is abundant, glycolysis will predominate. When glucose is scarce, gluconeogenesis will take over. F-2,6-BP inhibits F-1,6-BP which stops gluconeogenesis. At the same time, F-2,6-BP stimulates PFK which stimulates pyruvate kinase (promotes glycolysis). The key regulation site in the gluconeogenesis pathway is the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate (FIGURE 17.6). Consider first a situation in which energy is needed. In this case, the concentration of AMP is high. Under this condition, AMP stimulates phosphofructokinase but inhibits fructose 1,6-bisphosphatase. Thus, glycolysis is turned on and gluconeogenesis is inhibited. Conversely, high levels of ATP and citrate indicate that the energy charge is high and that biosynthetic intermediates are abundant. ATP and citrate inhibit phosphofructokinase, whereas citrate activates fructose 1,6-bisphosphatase. Under these conditions, glycolysis is nearly switched off and gluconeogenesis is promoted. Why does citrate take part in this regulatory scheme? As we will see in Chapter 19, citrate reports on the status of the citric acid cycle, the primary pathway for oxidizing fuels in the presence of oxygen. High levels of citrate indicate an energy-rich situation and the presence of precursors for biosynthesis, most notably fatty acid synthesis. * FIGURE 17.6 The reciprocal regulation of gluconeogenesis and glycolysis in the liver. The level of fructose 2,6-bisphosphate (F-2,6-BP) is high in the fed state and low in starvation. Another important control is the inhibition of pyruvate kinase by phosphorylation during starvation. Glycolysis and gluconeogenesis are also reciprocally regulated at the interconversion of phosphoenolpyruvate and pyruvate in the liver. The glycolytic enzyme pyruvate kinase is inhibited by allosteric effectors ATP and alanine, which signal that the energy charge is high and that building blocks are abundant. Conversely, pyruvate carboxylase, which catalyzes the first step in gluconeogenesis from pyruvate, is inhibited by ADP. Likewise, ADP inhibits phosphoenolpyruvate carboxykinase. Pyruvate carboxylase is activated by acetyl CoA, which, like citrate, indicates that the citric acid cycle is producing energy and biosynthetic intermediates (Chapter 19). Hence, when the cell is rich in biosynthetic precursors and ATP, anabolic pathways are favored. In the liver, gluconeogenesis can produce glucose to replenish glycogen stores (Chapter 25) and fatty acid synthesis can take place. In the liver, rates of glycolysis and gluconeogenesis are adjusted to maintain blood-glucose concentration. Recall that fructose 2,6-bisphosphate is a potent activator of phosphofructokinase (PFK), the primary regulatory enzyme in glycolysis (p. 329). Fructose 2,6-bisphosphate is also an inhibitor of fructose 1,6-bisphosphatase. When blood glucose concentration is low, fructose 2,6-bisphosphate is dephosphorylated to form fructose 6-phosphate, which no longer activates PFK. How is the amount of fructose 2,6-bisphosphate controlled to rise and fall with blood-glucose concentration? Two enzymes regulate the concentration of this molecule: one phosphorylates fructose 6-phosphate and the other dephosphorylates fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is formed from fructose 6-phosphate in a reaction catalyzed by phosphofructokinase 2 (PFK2), a different enzyme from phosphofructokinase. In the reverse direction, fructose 6-phosphate is formed through the hydrolysis of fructose 2,6-bisphosphate by a specific phosphatase, fructose bisphosphatase 2 (FBPase2). The striking finding is that both PFK2 and FBPase2 are present in a single 55-kDa polypeptide chain (FIGURE 17.7). This bifunctional enzyme contains an N-terminal regulatory domain followed by a kinase domain and a phosphatase domain. What controls whether PFK2 or FBPase2 dominates the bifunctional enzyme's activities in the liver? The activities of PFK2 and FBPase2 are reciprocally controlled by the phosphorylation of a single serine residue. When glucose is scarce, as it is during a night's fast, a rise in the blood concentration of the hormone glucagon triggers a cyclic AMP signal cascade (p. 248), leading to the phosphorylation of this bifunctional enzyme by protein kinase A (FIGURE 17.8). This covalent modification activates FBPase2 and inhibits PFK2, lowering the concentration of F-2,6-BP. Gluconeogenesis predominates. Glucose formed by the liver under these conditions is essential for the viability of the brain. Glucagon stimulation of protein kinase A also inactivates pyruvate kinase in the liver (p. 331). Conversely, when blood glucose is abundant, as it is after a meal, glucagon concentration in the blood falls and the insulin concentration rises. Now gluconeogenesis is not needed and the phosphoryl group is removed from the bifunctional enzyme. This covalent modification activates PFK2 and inhibits FBPase2. The resulting increase in the concentration of F-2,6-BP accelerates glycolysis. The coordinated control of glycolysis and gluconeogenesis is facilitated by the location of the kinase and phosphatase domains on the same polypeptide chain as the regulatory domain.

Understand the regulation of glycolysis via the following allosteric enzymes: hexokinase, phosphofructokinase (PFK), and pyruvate kinase. Separate your descriptions in the muscle vs. liver.

* The muscle is worried about itself, the liver is worried about the full body, making the liver more complex. The steps using enzymes Hexokinase, Phosphofructokinase (PFK), and Pyruvate Kinase all have -ΔG values (essentially irreversible reactions). The three steps with each of these enzymes are important steps that are regulated. The cell needs to make sure it wants the reaction to take place before it happens (because once the reaction starts, there's no going back due to the large -ΔG value). Regulation of Glycolysis in the Muscle Allosteric enzymes that catalyze steps with large -ΔG values: (1) Hexokinase: feedback inhibition (G-6P) (G-6P, the product, acts as an allosteric inhibitor. It binds to the allosteric site on hexokinase and will turn it off). - inhibition of PFK leads to inhibition of hexokinase (because the concentration of G6P will build up and inhibit hexokinase). (3) PFK: inhibited by high energy charge (you can slow down ATP-producing reactions when you don't need ATP), and low pH (due to lactic acid in muscles that are functioning anaerobically. Lactic acid decreases pH. Your cells are saying, "Hey, we can't let the pH of our cells continue to decrease, let's slow down glycolysis". - stimulated by low energy change (AMP) - committed step of glycolysis (most important control site in glycolysis in mammals)! (10) Pyruvate Kinase: inhibited by high energy charge, ATP (plenty of ATP) - stimulated by Fructose-1,6-BP (feed-forward stimulation) PFK sets the pace of cycle. Increasing levels of F-6P = high levels of ATP inhibit the enzyme by decreasing its affinity for F-6P. AMP will diminish that inhibitory effect of ATP = higher reaction velocity. ATP acts as an inhibitor (allosteric regulation). Muscle at Rest * Glycolysis inhibited. * ATP not needed, -> slow down glycolysis. * Hexokinase is inhibited by G-6P (negative feedback). * High-energy charge inhibits PFK and pyruvate kinase. Muscle during Exercise * Low energy charge or high levels of AMP will serve to stimulate PFK. The product of the reaction catalyzed by PFK gives Fructose-1,6-Bisphosphate, that'll stimulate pyruvate kinase. * ATP/AMP are allosteric regulators (directly binding to the enzyme PFK) * In exercising muscle, glycolysis is inhibited by excess ATP. Regulation of Glycolysis in Liver * The liver maintains blood glucose levels w/ large glycogen stores; a supplier tissue. (1) Hexokinase: in the liver isozyme glucokinase used. Isozyme = have slight amino acid differences. Hexokinase is called glucokinase and is also called hexokinase isozyme IV, they are all referring to the same thing. * Low affinity for glucose in liver, so it can give brain and muscle first call for glucose. * The liver takes in/stores glucose after meals, when [glucose] is high. * Not inhibited by G-6P (no feedback inhibition). * G-6P accumulates for synthesis of glycogen and fatty acids. Rather than G-6P proceeding through glycolysis, it'll be siphoned off for another purpose. * Inhibited when [F-6P] is high. (3) PFK: * inhibited by high [ATP]; stimulated by high [AMP] (energy charge) (ATP is less important because energy levels don't fluctuate in the liver the way they do in the muscle). * inhibited by citrate because citrate signals abundance in biosynthetic precursors <- so, it's saying that the citric acid cycle is full, we're doing fine, we have plenty of ways to get ATP, we can slow down; citrate is formed by step 1 CAC. * Stimulated by F-2,6-BP; glycolysis accelerated when glucose is abundant; feed-forward stimulation. * Not inhibited by low pH (lactate not normally produced in liver). (10) Pyruvate kinase: liver isozyme L form (not M form) * Isozymes differ in susceptibility to covalent modification ℗ (how susceptible they are to phosphorylation). * Inhibited when glucose levels are low via hormone signaling. * Inhibited also by high [ATP]. * F-6P is made during glycolysis. * F-2,6-BP is a signal molecule. It activates PFK, and the activation is required for PFK function - activating PFK increases PFK's affinity for F-6P and it'll diminish the inhibitory effect of ATP. * F-6P can only continue to proceed through glycolysis if PFK is active. However, PFK will only be activated if there is also F-2,6-BP available. * F-6P is converted to F-2,6-BP by PFK2. * Green is active. - Dephosphorylated in order to be active. - When phosphorylated -> less active. * Glucagon (opposite of insulin), "Glucose All Gone", is produced when glucose levels decrease. Glucagon is a hormone. * Glucagon initiates a signal transduction pathway that leads to PKA activation. PKA is a kinase that phosphorylates many target proteins including pyruvate kinase. We turn pyruvate kinase into a less active form when glucose levels are low, "If we don't have a lot of glucose, let's slow down glycolysis" * What would lead pyruvate kinase back into a dephosphorylated and more active state? A different signal transduction pathway that will activate phosphatase, an enzyme that will remove that phosphate group. * Feed-forward stimulation of F-1,6-BP: F-1,6-BP is an allosteric activator of pyruvate kinase. ATP and alanine are allosteric inhibitors. Pyruvate and ammonia combine to form alanine, so it's our signal that we have plenty of pyruvate, let's slow things down. The glycolytic pathway has a dual role: it degrades glucose to generate ATP, and it provides building blocks for biosynthetic reactions such as the formation of fatty acids and amino acids. The rate of conversion of glucose into pyruvate is regulated to meet these two major cellular needs. In metabolic pathways, enzymes catalyzing irreversible reactions are potential sites of control. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are irreversible, and each of them serves as a control site. These enzymes become more or less active in response to the reversible binding of allosteric effectors or covalent modification. We will consider the control of glycolysis in two different tissues—skeletal muscle and liver. Glycolysis in skeletal muscle provides ATP primarily to power contraction. Consequently, the primary control of muscle glycolysis is the energy charge of the cell—the ratio of ATP to AMP. Glycolysis is stimulated as the energy charge falls—a signal that the cell needs more ATP. Let's examine how each of the key regulatory enzymes responds to changes in the amounts of ATP and AMP present in the cell. Phosphofructokinaseis the most important control site in the mammalian glycolytic pathway. High levels of ATP allosterically inhibit the enzyme (a 340-kDa tetramer). ATP binds to a specific regulatory site that is distinct from the catalytic site. The binding of ATP lowers the enzyme's affinity for fructose 6-phosphate. AMP reverses the inhibitory action of ATP. AMP competes with ATP for the binding site but, when bound, does not inhibit the enzyme. Consequently, the activity of the enzyme increases when the ATP/AMP ratio is lowered (FIGURE 16.12). A decrease in pH also inhibits phosphofructokinase activity by augmenting the inhibitory effect of ATP. The pH might fall when fast-twitch muscle is functioning anaerobically, producing excessive quantities of lactic acid. The inhibition of glycolysis, and therefore of lactic acid fermentation, protects the muscle from damage that would result from the accumulation of too much acid. * FIGURE 16.12 The allosteric regulation of phosphofructokinase. A high level of ATP inhibits the enzyme by decreasing its affinity for fructose 6-phosphate. AMP diminishes the inhibitory effect of ATP. Why does AMP but not ADP stimulate the activity of phosphofructokinase? When ATP is being utilized rapidly, the enzyme adenylate kinase can form ATP from ADP by the following reaction: ADP + ADP ⇌ ATP + AMP Thus, some ATP is salvaged from ADP, and AMP becomes the signal for the low-energy state. Phosphofructokinase is the primary regulatory enzyme in glycolysis, but it is not the only one. Hexokinase, the enzyme catalyzing the first step of glycolysis, is inhibited by its product, glucose 6-phosphate. High concentrations of glucose 6-phosphate signal that the cell no longer requires glucose for energy, so no more glucose needs to be broken down. The glucose will then be left in the blood. A rise in glucose 6-phosphate concentration is a means by which phosphofructokinase communicates with hexokinase. When phosphofructokinase is inactive, the concentration of fructose 6-phosphate rises. In turn, the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-phosphate. Hence, the inhibition of phosphofructokinase leads to the inhibition of hexokinase. Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis? The reason becomes evident upon noting that glucose 6-phosphate is not solely a glycolytic intermediate. In muscle, for example, glucose 6-phosphate can also be converted into glycogen. The first irreversible reaction unique to the glycolytic pathway, the committed step (p. 128), is the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Thus, phosphofructokinase as the primary control site in glycolysis is highly appropriate. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway because it regulates flux down the pathway. Pyruvate kinase, the enzyme catalyzing the third irreversible step in glycolysis, controls the efflux from this pathway. This final step yields ATP and pyruvate, a central metabolic intermediate that can be oxidized further or used as a building block. ATP allosterically inhibits pyruvate kinase to decrease the rate of glycolysis when the energy charge of the cell is high. When the pace of glycolysis increases, fructose 1,6-bisphosphate, the product of the preceding irreversible step in glycolysis, activates the kinase to enable it to keep pace with the oncoming high flux of intermediates. A summary of the regulation of glycolysis in resting and active muscle is shown in FIGURE 16.13. * FIGURE 16.13 The regulation of glycolysis in muscle. At rest (left), glycolysis is not very active (thin arrows). The high concentration of ATP inhibits phosphofructokinase (PFK) and pyruvate kinase, while glucose 6-phosphate inhibits hexokinase. Glucose 6-phosphate is converted into glycogen (Chapter 25). During exercise (right), the decrease in the ATP/AMP ratio resulting from muscle contraction activates phosphofructokinase and hence glycolysis. The flux down the pathway is increased, as represented by the thick arrows. The liver has a greater diversity of biochemical functions than does muscle. Significantly, the liver maintains blood-glucose concentration: it stores glucose as glycogen when glucose is plentiful, and it releases glucose when supplies are low. It also uses glucose to generate reducing power for biosynthesis (Chapter 26) as well as to synthesize a host of building blocks for other biomolecules. So although the liver has many of the regulatory features of muscle glycolysis, the regulation of glycolysis in the liver is more complex. Liver phosphofructokinase can be regulated by ATP as in muscle, but such regulation is not as important since the liver does not experience the sudden ATP needs that a contracting muscle does. Likewise, low pH is not a metabolic signal for the liver enzyme, because lactate is not normally produced in the liver. Indeed, as we will see, lactate is converted into glucose in the liver. Glycolysis in the liver furnishes carbon skeletons for biosyntheses, so a signal indicating whether building blocks are abundant or scarce should also regulate phosphofructokinase. In the liver, phosphofructokinase is inhibited by citrate, an early intermediate in the citric acid cycle (Chapter 19). A high level of citrate in the cytoplasm means that biosynthetic precursors are abundant, so there is no need to degrade additional glucose for this purpose. In this way, citrate enhances the inhibitory effect of ATP on phosphofructokinase. The key means by which glycolysis in the liver responds to changes in blood glucose is through the signal molecule fructose 2,6-bisphosphate (F-2,6-BP), a potent activator of phosphofructokinase. After a meal rich in carbohydrates, the concentration of glucose in the blood rises. In the liver, the concentration of fructose 6-phosphate rises when blood-glucose concentration is high because of the action of hexokinase and phosphoglucose isomerase, and the abundance of fructose 6-phosphate accelerates the synthesis of F-2,6-BP (FIGURE 16.14). Hence, an abundance of fructose 6-phosphate leads to a higher concentration of F-2,6-BP. Fructose 2,6-bisphosphate stimulates glycolysis by increasing phosphofructokinase's affinity for fructose 6-phosphate and diminishing the inhibitory effect of ATP (FIGURE 16.15). Glycolysis is thus accelerated when glucose is abundant. Such a process is called feedforward stimulation. We will examine the synthesis and degradation of this regulatory molecule after we have considered gluconeogenesis (Chapter 17). * FIGURE 16.14 The regulation of phosphofructokinase by fructose 2,6-bisphosphate. High concentrations of fructose 6-phosphate (F-6P) activate the enzyme phosphofructokinase (PFK) through an intermediary, fructose 2,6-bisphosphate (F-2,6-BP). In the liver as well as in muscle, hexokinase is a regulatory enzyme. The hexokinase reaction is controlled in the liver as in muscle. However, the enzyme primarily responsible for phosphorylating glucose in the liver is not hexokinase, but glucokinase (hexokinase IV), an isozyme of hexokinase. Isozymes, or isoenzymes, are enzymes encoded by different genes with different amino acid sequences, yet they catalyze the same reaction. Isozymes usually differ in kinetic or regulatory properties. Glucokinase phosphorylates glucose only when glucose is abundant, as would be the case after a meal. The reason is that glucokinase's �M for glucose is about 50-fold higher than that of hexokinase, which means that glucose 6-phosphate is formed only when glucose is abundant. Furthermore, glucokinase is not inhibited by its product, glucose 6-phosphate, as hexokinase is. Moreover, when glucose concentration is low, glucokinase is inhibited by the liver-specific glucokinase regulatory protein (GKRP), which sequesters the kinase in the nucleus until the glucose concentration increases. The low affinity of glucokinase for glucose gives the brain and muscles first call on glucose when its supply is limited, and it ensures that glucose will not be wasted when it is abundant. Drugs that activate liver glucokinase or disrupt its interaction with GKRP are being evaluated as a treatment for type 2 or insulin-insensitive diabetes. Glucokinase is also present in the � cells of the pancreas, where the increased formation of glucose 6-phosphate by glucokinase—when blood-glucose levels are elevated—leads to the secretion of the hormone insulin. Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat. Several isozymic forms of pyruvate kinase (a tetramer of 57-kDa subunits) encoded by different genes are present in mammals: the L form predominates in liver; the M form, in muscle and brain. The L and M forms of pyruvate kinase have many properties in common. Indeed, the liver enzyme behaves much as the muscle enzyme does in regard to allosteric regulation, except that the liver enzyme is also inhibited by alanine (synthesized in one step from pyruvate), a signal that building blocks are available. Moreover, the isozymic forms differ in their susceptibility to phosphorylation. The catalytic properties of the L form—but not of the M form—are controlled by reversible phosphorylation (FIGURE 16.16). When the blood-glucose concentration is low, the glucagon-triggered cyclic AMP cascade (p. 351) leads to the phosphorylation of pyruvate kinase, which diminishes its activity. This hormone-triggered phosphorylation prevents the liver from consuming glucose when it is more urgently needed by brain and muscle. We see here a clear-cut example of how isoenzymes contribute to the metabolic diversity of different organs. We will return to the control of glycolysis after considering gluconeogenesis (Chapter 17). * FIGURE 16.16 The control of the catalytic activity of pyruvate kinase. Pyruvate kinase is regulated by allosteric effectors and covalent modification. Fructose 1,6-bisphosphate allosterically stimulates the enzyme, while ATP and alanine (in liver) are allosteric inhibitors. Glucagon, secreted in response to low blood glucose, promotes phosphorylation and inhibition of the enzyme. When blood-glucose concentration is adequate, the enzyme is dephosphorylated and activated.

Be familiar with the epinephrine signaling pathway discussed: (1) activation of adenylate cyclase & (2) activation of phospholipase C. Be able to sketch the pathway.

.

Know this table.

.

Place an "X" in the appropriate box that leads to an increase in activity of that pathway:

.

List 5 secondary messengers that are commonly used in animal cells.

1. Cyclic AMP (cAMP) 2. Cyclic GMP (cGMP) 3. Ca2+ 4. IP3 5. Diacyglycerol (DAG)

What are the 3 classes of membrane receptors? Understand their main features, mechanism of action, examples, and how the signal is terminated. First Type

1. Seven-Transmembrane-Helix Receptors 2. Dimeric receptors that recruit protein kinases 3. Dimeric Receptors that are protein Kinases 1. 7TM Helix Receptors (β-adrenergic receptor) * β-adrenergic receptor binds epinephrine. * 7TM Helix Receptors are comprised of 7 alpha helices that pass through the cell membrane. * Epinephrine binds to the 7TM receptor known as the β-adrenergic receptor, and after signal transduction has taken place, the different cellular responses (fight or flight responses) include heart muscle contraction, divert blood flow to skeletal muscle, glycogen breakdown, and more. * Ligand binding causes conformational change in the receptor. * 7 alpha helices each spans the membrane, common receptor motif. Epinephrine binding to that 7TM receptor. When it does so, that receptor changes its conformation. When it changes its conformation (shape), its going to activate the G-protein. This G protein has 3 parts, making it a heterotrimeric G protein. Notice it has α, β, γ subunits. That conformational change in its cytoplasmic domain (highlighted in pink) of the 7TM receptor will activate the G protein. Specifically, the receptor triggers an exchange of GDP (guanosine diphosphate) to GTP (guanosine triphosphate). Additionally, there's a dissociation of the β and γ subunits. Highlighted in yellow is the activated α subunit of this G protein. NOTED: Its not phosphorylation at this point, just an exchange of GDP for GTP. GTP, an activated G protein, will activate the orange enzyme known as Adenylate Cyclase, AKA Adenolil Cyclase. This enzyme catalyzes the formation of Cyclic AMP from ATP, just like the name suggests (cAMP). Cyclic AMP (our secondary messenger) activates a kinase (protein kinase A or pKa). Binding of cyclic AMP to pKa activates it. * Adenylate cyclase converts ATP to cAMP and cAMP will activate pKa. * Kinases phosphorylate substrates. * Upon inactive heterotrimeric G protein binding to the hormone receptor complex (epinephrine bound to the β-adrenergic receptor), GDP is released and GTP binds to that α subunit. It is now in its active form and binds to and activates adenylate cyclase. We don't want the active G protein to stay active forever, we want to turn it off. That happens through GTPase activity. - GTPase activity is not due to a separate enzyme. Instead, the α subunit has intrinsic GTPase activity. It will hydrolyze the bound GTP to GDP plus an organic phosphate, and now it is inactive and will reassociate with the β and γ subunits. * Adenylate cyclase has 12 membrane spanning helices. * Gαs: In the active form of the G protein (α subunit bound to GTP form), s = stimulatory. Gαs stimulates adenylate cyclase. In order to activate pKa, four individual cAMP molecules need to bind to the cAMP binding domains. When that occurs. the 2 regulatory chains will dissociate from pKa, and now you have the C catalytic subunits which become active. These active molecules phosphorylate other proteins, specifically Serine and Threonine residues. 7TM-GPCR: another way we can organize 7TM receptors is by those that subsequently activated a G protein. We call these the 7TM G-protein coupled receptors. The adrenergic receptor can activate other pathways as well. A 7TM-GPCR activates 2 major pathways: 1. Gαs -> adenylate cyclase -> cAMP -> pKa * breaks down glycogen for fuel and halts synthesis of glycogen. Once pKa is activated, glycogen will be broken down for fuel. The synthesis for the storing of glucose in the form of glycogen is going to be halted. This is because when epinephrine activates the β-adrenergic receptor, you have a fight or flight response. Glycogen will be broken down so that we can provide energy for muscle contraction and so forth. 2. Gαq -> Phospholipase C (PLC) -> DAG + IP3 * PLC -> DAG -> pKc * PLC -> IP3 -> Ca2+ -> pKc and/or calmodulin binding * Calmodulin (with Ca2+) -> CaM kinase - breaks down glycogen for fuel and halts synthesis of glycogen. In addition to Gα subunit becoming stimulated (Gαs), other hormones can bind to this receptor and activate a G protein that activates a different enzyme called phospholipase C (PLC). This G protein is called Gαq. Once PLC is activated, two additional second messengers are activated: DAG and IP3. DAG will go on to activate a protein kinase known as protein kinase C (pKc). IP3 will stimulate a calcium release that then can also serve to activate pKc, or it will activate a kinase called CaM kinase (calmodulin kinase). This is a different pathway entirely from the first, but you can see the outcome is the same. We break down glycogen as fuel and we're going to stop storing up glucose in the form of glycogen, we're going to stop glycogen synthesis. * PLC Pathway: A messenger such as vasopressin will bind to the 7TM receptor that will activate the heterotrimeric G-Protein. In doing so, GDP is exchanged for GTP. That αq subunit will activate the enzyme PLC. PLC cleaves the phospholipid PIP2 into two products: Diacylglycerol (DAG) and IP3. DAG will activate protein kinase C, which phosphorylates other proteins will IP3 will bind to receptors in the endoplasmic reticulum. These receptors will open up and cause a release of calcium into the cytosol. Calcium will bind to calmodulin, which will activate protein kinase C, and there are other responses as well such as contraction and secretion. The purple receptor is called the IP3 receptor. * PLC is in the plasma membrane. * DAG stays within the membrane * IP3 can diffuse into the cytoplasm Upon pKc activation, it phosphorylates Serine and Threonine residues. Calmodulin is activated by Ca2+ binding. CaM is a calcium sensor. Activated CaM binds (and activates) CaM kinase and other proteins (CaM kinase is a calmodulin-dependent protein kinase). CaM kinase also adds PO4 to glycogen synthase to inactivate it. * Once IP3 receptor is activated by IP3, calcium will flow from the endoplasmic reticulum out into the cytoplasm. Those high levels of calcium will trigger a variety of biochemical processes such as smooth muscle contraction and glycogen breakdown. * Once calcium is out into the cytoplasm, it can bind to a number of molecules including CaM. All proteins in the EF hand family bind to Ca2+. Activating CaM kinase enables this kinase to go on and phosphorylate a wide variety of target proteins. There are other targets for CaM, not just CaM kinase. Calcium is important in signal transduction. * Calcium is a second messenger localized in space and time. Ending Signaling * G protein "turns off" as GTP -> GDP * cAMP broken down (remove 2nd messenger) by cAMP phosphodiesterase. * Ca2+ pumped out of the cell or stored in the ER. * IP3 degraded or phosphorylated. * DAG converted to phosphotidate or hydrolyzed. * Ligand dissociates from the receptor. * Receptor can be removed from the membrane; sequestered into intracellular vesicles. Signal termination 1 - Gα Protein * GTP (G protein in active form) uses GTPase activity to convert GTP back to GDP, and we have reassociation with the β and γ subunits. Signal termination 2 - at the receptor * Dissociation of epinephrine from the receptor. * Without the epinephrine, the cytoplasmic domain will no longer activate a G protein. More than 700 GPCR's in humans 50% of drugs target different GPCR's

What are the 3 classes of membrane receptors? Understand their main features, mechanism of action, examples, and how the signal is terminated. Second Type

1. Seven-Transmembrane-Helix Receptors 2. Dimeric receptors that recruit protein kinases 3. Dimeric Receptors that are protein Kinases 2. Dimeric Receptors That Recruit Protein Kinases (Human Growth Hormone Receptor) * Extracellular domains of two HGH receptors bind to one growth hormone. * The ligand is the growth hormone. * Ligand binding changes quaternary structure of the receptor, results in receptor dimerization. When the two receptors come together, they are now activated. JAK2 associates with the intracellular domain of the receptor. * Dimerization brings two JAK2 proteins together. * Each of these kinases will phosphorylate their partner. - JAK2 will phosphorylate tyrosine residues on the other kinase. * Activated JAK2 will phosphorylate a transcription factor known as STAT. - STAT will go to the nucleus and will bind to DNA and regulate gene expression. SUMMARY: The HGH (ligand) binds to the HGH receptor, leading to dimerization of the receptor and the activation of JAK2. THe JAK2's will cross phosphorylate eachother, leading to the activation of STAT5. STAT5 is a transcription factor. Once it's phosphorylated, it can move to the nucleus, bind to DNA, and control transcription. ANOTHER SUMMARY: Hormone binds -> Receptor dimerization -> cross-phosphorylation of JAK2 -> JAK2 phosphorylates other targets -> ex. JAK2 phosphorylates STAT5 and STAT 5 binds to DNA to activate transcription. * You can have other signal proteins that bind to receptors and lead to STAT activation. Human growth hormone receptor: JAK2 -> STAT----------------------------------The signal for this type is terminated through the use of intrinsic GTPase activity, which is used to turn the signal off.

What are the 3 classes of membrane receptors? Understand their main features, mechanism of action, examples, and how the signal is terminated. Third Type

1. Seven-Transmembrane-Helix Receptors 2. Dimeric receptors that recruit protein kinases 3. Dimeric Receptors that are protein Kinases 3. Dimeric Receptors That Are Protein Kinases * The receptor itself is a tyrosine kinase. * Previously, tyrosine kinase was recruited. In this case, the receptor is already a tyrosine kinase. * Two ligands will bind, each to their own receptor. This leads to dimerization. Then we get cross-phosphorylation (autophosphorylation). EGF Receptor is a receptor tyrosine kinase. During autophosphorylation, the kinase domains of these intracellular domains will phosphorylate one another. Again, this is ligand-induced dimerization, and each kinase domain will catalyze the phosphorylation of its partner. After cross phosphorylation, the phosphorylated receptor will bind to the purple protein known as Grb-2. That binds to the blue protein known as Sos. That stimulates the exchange of GTP for GDP in this G protein, known as Ras. Now that it is bound to GTP, its activated, and the signal continues. Activated Ras will bind and activate other protein kinases. * There are many receptor tyrosine kinases. EGF Receptor Domains: * Ligand binds -> Receptor dimerization. * 2 receptor kinase domains will phosphorylate eachother making each active (cross-phosphorylation of Tyr residues). * Once activate, they bind adapter proteins: Grb2. * Protein-protein interactions transmit the signal. * G-protein Ras is activated, Ras continues signal. * The EGF binding domain is the portion of the receptor that's on the cell surface. * The kinase domain is the portion in the cytoplasm. * The C-terminal tail is tyrosine rich and the area that will be phosphorylated through cross-phosphorylation. Ras belongs to a larger family of G proteins called the Ras Superfamily. Ras is a monomeric GTPase, a molecular switch. This is different from the heterotrimeric which we learned about previously with the β-adrenergic receptor pathway. Other than that, the pattern is the same. Ras is a G-Protein, it is inactive when bound to GDP, interaction with Sos will stimulate the exchange of GTP for GDP, once bound to GTP, Ras is now active and it will activate Serine/Threonine kinases. Once again, intrinsic GTPase activity results in a return of that GTP back to GDP. Ras's intrinsic GTPase activity serves to terminate the signal and return the system to the inactive state. NOTE: We're not just popping GDP in and out. We're removing the entire nucleotide during the activation step. But when we return back to the GDP bound form, that's through a different mechanism. That's through GTPase activity. EGF Receptor: Grb -> Sos -> Ras -> kinases activated

What is F-2,6-BP?

A glycolysis activator.

Define and differentiate a primary messenger and second messenger.

A stimulus such as a wound or a digested meal triggers the release of the signal molecule (ligand), also called the primary messenger. Structural changes in receptors lead to changes in the concentration of small molecules, called second messengers, that are used to relay information from the receptor-ligand complex. Summary: primary messenger is the signaling molecule while the second messenger relays information, often amplifying the message. 1. Signal molecule (primary messenger, ligand) binds cell receptor. 2. Cell receptor changes conformation and triggers intracellular second messenger molecule. 3. Second messenger activates intracellular responses (often amplifies the message). 4. Activation of effectors that alter the physiological response - Changes in protein activity or gene expression. 5. Termination of the signal Most signal molecules (primary receptors) are too large and too polar to pass through the cell membrane or through transporters. Thus, the info presented by signal molecules must be transmitted across the cell membrane without the molecules themselves entering the cell. Membrane receptors transfer info from the environment to a cell's interior. Such receptors are integralmembrane proteins that have both extracellular and intracellular domains. A binding site on the extracellular domain specifically recognizes the signal molecule (ligand, primary messenger). The formation of the receptor-ligand complex alters the tertiary or quaternary structure of the receptor, including the intracellular domain. However, structural changes in the few receptors that are bound to ligands are not sufficient to yield a response from the cell. The info conveyed by the receptor must be transduced into other forms of info that can alter the cell's biochemisty. Structural changes in receptors lead to changes in the concentration of small molecules, called second messengers, that are used to relay info from the receptor-ligand complex. The use of second messengers have several consequences. One consequence is that second messengers are often free to diffuse to other compartments of the cell, such as the nucleus, where they can influence gene expression and other processes. Another consequence is that the signal may be amplified significantly in the generation of second messengers. Each activated receptor-ligand complex can lead to the generation of many second messengers within the cell. Thus, a low concentration of signal molecules in the environment, even as little as a single molecule, can yield a large intracellular signal and response. Second messengers relay info from the receptor-ligand complex into the cell. These are the intracellular molecules that will change in concentration in response to environmental signals. They're small so they can easily diffuse into other compartments, like the nucleus. They also assist in that amplification. You can have one ligand binding to the receptor, generating many second messenger molecules.

Be familiar with coenzymes common in metabolism and what they carry: NAD+, FAD, CoA, NADP+.

Activated Carriers of Electrons for Fuel Oxidation * NAD+ can carry: H- (hydride ion, 1 proton and 2 e-) * FAD can carry: 2 H's (2 protons and 2 e-) NAD+ is an e- carrier. Here, we have NAD+ in the oxidized form. The nicotinamide ring will accept a hydrogen ion and two e-, to give us NADH (the reduced form). NAD+ is a great e- acceptor. So, in this example, we have a proton and two e- directly transferred to NAD+, and then the other proton appears in the solvent. FAD is another great e- carrier. Just like NAD+, it can also accept 2 e-. However, it takes up two protons as well, unlike NAD+. Here, we see FAD in the oxidized form. In the reduced form, FADH2, two protons have been taken up along with 2 e-. Activated Carriers of Electrons for the Synthesis of Biomolecules Products are more reduced than the reactants. NADPH is the electron donor used (NADH + phosphate) * NADPH used for reductive biosynthesis * NADH used for the generation of ATP We need high potential e- to be used in anabolic processes, compared to catabolic processes. Because the precursors are more reduced than the products. So we need some reducing power in addition to ATP. So, as an example, in the biosynthesis of fatty acids, the keto group is reduced to a methylene group, and that actually requires the input of 4 e-. * NADP+ is the oxidized form. NADPH will carry e- in the same way as NADH did. * CoA-SH (Coenzyme A) carries an acyl group. Important in catabolism and synthesis of fatty acids. Acyl group is linked by a thioster bond that is a 'high energy' bond due to it not being stabilized by resonance (hydrolysis is favored). Bond has a high acyl-group-transfer potential. CoA is a carrier of two carbon fragments. We need acyl groups in the oxidation of fatty acids and for the synthesis of membrane lipids. The hydrolysis of a thioester bond is thermodynamically more favorable than that of an oxygen ester. NAD+, FAD, and NADP+ are all e- carries. CoA carries 2C fragments/acyl groups.

Differentiate between aerobic respiration and anaerobic respiration.

Anaerobic and Aerobic Catabolism of Glucose Provides Energy for: 1. Mechanical work 2. Active transport 3. Synthesis of macromolecules While aerobic metabolism generates more ATP and relies on oxygen, anaerobic metabolism does not need oxygen but only creates two ATP molecules per glucose molecule. However, anaerobic and aerobic metabolism are both required to produce cellular energy.

Why are vitamins necessary for metabolism and good health?

Because many of the activated carriers are coenzymes that are derived from water-soluble vitamins. Activated carriers exemplify the modular design and economy of metabolism. ATP is an activated carrier of phosphoryl groups. - Transfer of the phosphoryl group from ATP is energetically favorable (exergonic, negative ΔG) Coenzymes are often activated carriers. Coenzymes carry electrons removed by oxidation of fuels (in glycolysis and the citric acid cycle) - Coenzymes have a higher affinity for e-'s than C fuels, but lower affinity for e-'s than O2.

Explain why glucose is usually not the final product of gluconeogenesis.

Because of Bypass step #2. In most tissues, except the liver, F-6P -> G-6P. In the liver, G-6P can still be converted into glucose. In most tissues, gluconeogenesis ends with the formation of glucose 6-phosphate. Free glucose is not generated because most tissues lack glucose 6-phosphatase. Rather, the glucose 6-phosphate is commonly converted into glycogen, the storage form of glucose. It is really important to take note of figure 17.5, that the enzyme needed to convert G6P into glucose is primarily produced in the liver, and not in other tissues around the body. In this way, the liver serves to control release of glucose into the blood.

What are the bypass steps of gluconeogenesis? Be able to match the enzymes with the reactions they catalyze.

Bypass step #1: * Pyruvate carboxylase uses ATP to add CO2 to pyruvate. * Pyruvate -> oxaloacetate -> phosphoenolpyruvate - carboxylation then decarboxylation. * Decarboxylation reactions are often coupled to reactions that are otherwise highly endergonic. - Adding a phosphate to pyruvate to convert pyruvate to PEP (highly unfavorable). - Reverse of glycolysis step ΔG°' = +31 kJ/mol (highly unfavorable). - Steps in gluconeogenesis ΔG°' = +0.8 kJ/mol (highly unfavorable). * Decarboxylation can drive otherwise highly endergonic reactions forward. Compartmentalization: Gluconeogenesis occurs in the cytoplasm. Pyruvate Carboxylase is in the mitochondria (#1). Oxaloacetate must be transported mitoch -> cytoplasm, but there's no oxaloacetate transporter so it first has to be converted into malate (there's a malate transporter). Malate is then reoxidized to oxaloacetate in the cytoplasm and oxidation now proceeds to the next step which is the conversion into PEP by PEP carboxykinase. Bypass Step #2 Gluconeogenesis: * Fructose 1,6-Bisphosphatase is an allosteric enzyme important for regulation of gluconeogenesis (it catalyzes the step right before what we would consider the end of gluconeogenesis, which is F-6P -> G-6P. * End of gluconeogenesis in most tissues (except liver), F-6P -> G-6P (phosphoglucose isomerase). In the liver, this is not the end, G-6P can still be converted into glucose. Bypass Step #3 Gluconeogenesis: Occurs mostly in the liver, glucose released. Compartmentalization in the ER. G-6P -> Glucose by G-6-Phosphatase. * Free glucose is not formed in the cytoplasm. The G-6P is transported by transporter T1 into the ER lumen, where it is hydrolyzed by G-6P, an enzyme that's bound to the ER membrane into an inorganic phosphate and glucose. - T2 will transport the inorganic phosphate and T3 will transport glucose back out to the cytoplasmic side.

Differentiate between catabolic reactions and anabolic reactions.

Catabolism: reactions that transform fuels into cellular energy; oxidation of glucose. Anabolism: reactions that require energy; synthesis of molecules. Each pathway is usually distinct: * 1 enzyme(s) used for key steps of a catabolic pathway and a different enzyme(s) used for key steps of an anabolic pathway. * Enhances control over these pathways. Cells and food molecules result in useful forms of energy, although some of that is lost as heat. We end up with the building blocks of biosynthesis. These would be catabolic pathways. Those building blocks used for biosynthesis will form many new molecules, but we have to have an input of energy. Those are anabolic reactions. Our cells continually need to make more ATP. ATP is used to power motion, active transport, biosynthesis, signal amplification.: Anabolic rxns. ADP is converted back into ATP through oxidation of fuel molecules or photosynthesis.

Citric Acid Cycle Mnemonics CAC Enzymes

Cathy Always Is Asking Saint Sebastian For More Citrate Synthase Aconitase Isocitrate dehydrogenase α-Ketoglutarate dehydrogenase complex Succinyl CoA synthetase Succinate dehydrogenase Fumarase Malate dehydrogenase

How is fructose altered for entry into glycolysis?

Fructose is converted into glycolytic intermediates. The sugar fructose doesn't have its own metabolic pathway. Instead, it is converted into intermediates along the glycolysis pathway (both fructose and galactose are converted into glycolytic intermediates in this way. Organisms can use other sugars besides glucose). * Fructose (adipose tissue) -> Fructose-6P (now entered into glycolysis pathway, and continues on down the pathway, producing GAP and DHAP by F-1,6-BP, etc.). In the liver, the pathway is different. * Most fructose is processed in the liver. - Fructose (liver) -> DHAP <-> GAP -> pyruvate (2x) 3-step pathway used by the liver, metabolizes most fructose. * DHAP converted to GAP by triose phosphate isomerase (glycolysis step #5). * GAP converted to 1,3-BPG (glycolysis step #6). Adipose tissue uses hexokinase to phosphorylate. * Fructose -> fructose-6-phosphate -> step 3 glycolysis. Fructose can take one of two pathways to enter the glycolytic pathway. Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway (FIGURE 16.8). The first step is the phosphorylation of fructose to fructose 1-phosphate by fructokinase. Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate, an intermediate in glycolysis. This aldol cleavage is catalyzed by a specific fructose 1-phosphate aldolase. Dihydroxyacetone phosphate continues into stage 2 of glycolysis, whereas glyceraldehyde is then phosphorylated to glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase. In other tissues such as adipose tissues, fructose can be phosphorylated to the glycolytic intermediate fructose 6-phosphate by hexokinase.

Explain the action of glyceraldehyde-3-phosphate dehydrogenase and the formation of the thioester intermediate.

GAP is converted into 1,3-BPG by the enzyme Glyceraldehyde-3-Phosphate Dehydrogenase (GAP DH). * This conversion is a redox reaction. * 1,3-BPG is an acyl-phosphate with high phosphoryl transfer potential. - The aldehyde on GAP is converted to a carboxylic acid on 1,3-BPG. The H from the aldehyde is transfered to NAD+ to generate NADH in the final product of this step. NADH is an important e- carrier. - We can also see the acyl-phosphate formation (dehydration). * The first step ΔG°' value is favorable while the second step ΔG°' value is unfavorable. We have to couple these reactions to drive the formation of an acyl phosphate. - If the reactions were not coupled, as we can see in the first graph, "Energy of 2 separate reactions", we would have a very large ΔG‡ (activation energy). - However, in reality, the reactions are coupled with a thioester intermediate, which is a cysteine residue in the active site that has a thiol group that reacts with the carbonyl group on GAP. The thioester intermediate is more stable than the reactant, and hence, its formation is spontaneous. But, the intermediate is less stable than the products, so, those will also form spontaneously. Let's consider this reaction in some detail because it illustrates the essence of energy transformation and metabolism itself: the energy of carbon oxidation is captured as high phosphoryl-transfer potential. The reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase can be viewed as the sum of two processes: the oxidation of the aldehyde (in this case, glyceraldehyde 3-phosphate) to a carboxylic acid by NAD+ and the joining of the carboxylic acid (3-phosphoglycerate) and orthophosphate to form the acyl-phosphate product, 1,3-bisphosphoglycerate. The first reaction is thermodynamically quite favorable, with a standard free-energy change, ΔG∘′, of approximately −50 kJ mol−1 (−12 kcal mol−1), whereas the second reaction is quite unfavorable, with a standard free-energy change of the same magnitude but the opposite sign. If these two reactions simply took place in succession, the second reaction would not take place at a biologically significant rate, because of its very large activation energy (FIGURE 16.3). These two processes must be coupled so that the favorable aldehyde oxidation can be used to drive the formation of the acyl phosphate. How are these reactions coupled? The key is an intermediate that is linked to the enzyme by a thioester after the aldehyde has been oxidized. This intermediate reacts with orthophosphate to form the high-energy compound 1,3-bisphosphoglycerate. The thioester is a free-energy intermediate between the aldehyde and the free carboxylic acid. The favorable oxidation and unfavorable phosphorylation reactions are coupled by the thioester intermediate, which preserves much of the free energy released in the oxidation reaction (FIGURE 16.3B). * FIGURE 16.3 Free-energy profiles for glyceraldehyde oxidation followed by acyl-phosphate formation. (A) A hypothetical case with no coupling between the two processes. The second step has a large activation barrier, making the reaction very slow. (B) The actual case with the two reactions coupled through a thioester intermediate. The thioester intermediate is more stable than the reactant, and hence, its formation is spontaneous. However, the intermediate is less stable than the product, which forms spontaneously. Thus, the barrier separating oxidation from acyl-phosphate formation is eliminated.

Understand the regulation of gluconeogenesis.

Gluconeogenesis and glycolysis are reciprocally regulated. - Via distinct enzymes at key steps. * When glucose is abundant - glycolysis active. * When [glucose] is low - gluconeogenesis is active. * Both are responsive to the energy charge of the cell. * Also responsive to the abundance of biosynthetic intermediates: citrate, alanine, and acetyl CoA (alanine and acetyl CoA are products of pyruvate). They are coordinated so that when one pathway is active, the other should be relatively inactive, vise versa. Reciprocal Regulation in the Liver * ATP inhibits PFK, and pyruvate kinase. * Citrate and low pH will inhibit PFK while alanine inhibits pyruvate kinase. * F-2,6-BP and low energy charge will stimulate PFK and F-1,6-BP stimulates pyruvate kinase. Conversion of pyruvate into oxaloacetate by pyruvate carboxylase. Pyruvate carboxylase is stimulated by acetyl CoA and inhibited by ADP. The enzyme PEP carboxy kinase is inhibited by ADP. The conversion of F-1,6-BP to F-6-P, citrate will stimulate F-1,6-BP, but F-2,6-BP and AMP will inhibit this enzyme. AMP is telling the cell, "we need energy, stimulate PFK but inhibit Fructose-1,6-bisphosphatase". * In the liver, glycolysis is inhibited by ATP while gluconeogenesis is inhibited by ADP. Glucose Metabolism and the Liver * The liver functions to maintain glucose levels in the blood. * Metabolism in the liver is affected by insulin and glucagon (both hormones). * Glucagon is released when glucose levels in the blood are low. - Glucagon signaling results in phosphorylation of PFK2 (by PKA) in the liver. - Inhibits glycolysis: removes F-2,6-BP (glycolysis activator) and results in phosphorylation of pyruvate kinase (making it inactive). Without F-2,6-BP we can't stimulate PFK. Without PFK, we can't proceed through glycolysis. - Stimulates gluconeogenesis: decreases inhibitor F-2,6-BP. (Produces from F-6P by enzyme PFK2. Glycolysis is used by the liver to generate intermediates for biosynthesis, not for ATP/energy production. PFK2 in the liver: In the liver, glucagon results in phosphorylation of PFK2 = stimulates gluconeogenesis. * PFK2 has two domains: kinase domain and the phosphatase domain. * Kinase domain: phosphorylates F-6P -> F-2,6-BP * Phosphatase domain: dephosphorylates F-2,6-BP -> F-6P - Since these have opposing activities, we can't have both domains active at the same time. We can control this through covalent modification. * FBPase2: phosphate activity. PKA leads to the phosphorylation and thereby the inactivation of PFK2 kinase activity. In doing so, phosphatase domain is active -> FBPase2, which removes a phosphoryl group from Fructose-2,6-BP and turns it back into F-6P. * Insulin leads to the activation of phosphoprotein phosphatase, which removes the phosphoryl group from PFK2. in the dephosphorylated form, the enzyme is called PFK2 and has kinase activity, leading to the phosphorylation of Fructose-6-phosphate to produce F-2,6-BP.

Memorization Mnemonics for Substrates of Glycolysis

Goodness (Glucose) Gracious (Glucose-6P) Father (Fructose-6P) Franklin (Fructose-1,6-BP) Did (Dihydroxyacetone-P) Go (Glyceraldehyde-3P) By (1,3-BPG) Picking (3-Phosphoglycerate) Pumpkins (2-Phosphoglycerate) (to) PrEPare (Phosphoenolpyruvate [PEP]) Pies (Pyruvate).

Memorization Mnemonics for Enzymes of Glycolysis

High (Hexokinase) Profile (Phosphoglucose isomerase) People (Phosphofructokinase) Act (Aldolase) Too (Triose phosphate isomerase) Glamorous (Glyceraldehyde-3-phosphate dehydrogenase Picture (Phospoglycerate kinase Posing (Phosphoglycerate mutase) Every (Enolase) Place (Pyruvate kinase)

At what step do glycerol, lactate, and amino acids enter gluconeogenesis?

Lactate and some amino acids enter at the first step. They are converted into pyruvate and then follow the regular steps of gluconeogenesis. Some amino acids enter at the second step. They are converted into oxaloacetate, and then to phosphoenolpyruvate by the enzyme phosphoenolpyruvate carboxykinase, etc. etc. (following the regular steps of gluconeogenesis). Glycerol enters midway into the pathway through DHAP. 2 glycerol -> 1 G-6P.

List 2 general responses that can result from signal transduction.

Signal transduction allows an organism to sense its environment and formulate the proper biochemical response. Signal transduction is how cells recieve, process, and respond to information from the environment whether the information is in the form of light, smell, or blood-glucose concentration. Signal transduction cascades mediate the sensing and processing of these stimuli. These molecular circuits detect, amplify, and integrate diverse external signals to generate responses such as changes in enzyme activity, gene expression, or ion-channel activity.

Match the enzyme on the right with the appropriate control mechanisms.

MATCH!

Reactions must be thermodynamically favorable in order to occur in cells. How are reactions with positive Delta G values made thermodynamically favorable?

Metabolism is composed of many interconnecting reactions. Metabolism consists of energy-yielding reactions and energy-requiring reactions. A thermodynamically unfavorable reaction can be driven by a favorable reaction. * (-) charged: favorable * (+) charged: unfavorable Metabolic pathways are formed by the coupling (adding together) of enzyme-catalyzed reactions that lead to a thermodynamically favorable reaction. * Negative ΔG values indicate energetically favorable/spontaneous. * Coupling reactions to the hydrolysis of ATP makes many reactions now favorable. ATP is an energy coupling agent. - We couple its hydrolysis to another reaction, and in doing so, we convert an unfavorable reaction -> favorable one. ATP is energy rich due to its two phosphoanhydride linkages. A large amount of energy is released when ATP is hydrolyzed to ADP, or to AMP. Compounds with high phosphoryl transfer potential can couple carbon oxidation to ATP synthesis. Carbon oxidation generates an acyl phosphate known as 1,3-BPG. The e- captured by NAD+ and 1,3-BPG has a high phosphoryl transfer potential. Cleavage of the 1,3-BPG is coupled to the synthesis of ATP. So, the energy of oxidation is initially trapped as the high phosphoryl transfer potential molecule (1,3-BPG), and then its used to form ATP. Phosphoryl transfer can be used to drive otherwise thermodynamically unfavorable reactions.

Understand what is meant by energy charge of the cell.

Metabolism is often controlled by the energy status of the cell. * Energy charge can range from 0 (all AMP) to 1 (all AMP). High energy charge inhibits catabolic reactions (= near 1). Low energy charge inhibits anabolic reactions (= near 0). If I have plenty of energy, why should a catabolic reaction take place? Let me inhibit glycolysis if I already have plenty of ATP. Anabolic reactions shouldn't take place if there isn't ATP available to power them. Relating to Practice Problem Recall energy charge is between 0-1. When the energy charge is low, catabolic reactions will be very active. However, as the energy charge increases within the cell, those catabolic reactions will slow down and will have an increase in anabolic reactions. The reactions that use energy will be active when that energy is available. When energy charge is low, we have very little ATP so catabolic reactions are in full gear. But, as we start to approach 1 on the x-axis, energy charge is very high, and we have lots of ATP. Now, anabolic reactions will be in full gear making glycogen, proteins, fatty acids, using ATP to do so. Catabolic reactions would be breaking down fatty acids, glucose, proteins, all to get ATP because clearly the cell needs ATP.

Citric Acid Cycle Mnemonics CAC Intermediates

Our City Is Kept Safe and Sound From Malice Oxaloacetate Citrate Isocitrate α-ketoglutarate Succinyl-coA Succinate Fumarate Malate

Compare molecules in a reaction and be able to determine which ones are oxidized vs. reduced over the course of a reaction.

Oxidized means loss of e- (less H/Increase in O) EX.: Gained a double bond Reduced is gain of electrons (more H/decrease in O) EX: Lost a double bond

Explain the possible fates of pyruvate.

Pyruvate is oxidized into Acetyl CoA, which is oxidized further during the Citric Acid Cycle or the Krebs Cycle. But, this is under aerobic conditions. The fate of pyruvate is variable. THESE (highlighted in pink) reactions do not require oxygen. The formation of lactate happens during lactic acid fermentation and the formation of ethanol occurs during alcohol fermentation.

** Not on SG ** Know the steps of glycolysis and the characteristics of each step. Be able to recognize structures.

STEP #1: Glucose is converted into Glucose 6-Phosphate by enzyme hexokinase. Hexokinase catalyses this reaction, and takes a phosphoryl group from ATP and adds that to glucose. The phosphoryl group traps glucose in the cell. G-6P cannot pass back out of the cell. The phosphoryl group also destabilizes glucose, and facilitates further metabolism. * Kinases add a phosphoryl group from ATP to a substrate and require Mg2+/Mn2+ as a cofactor. STEP #2: G-6P -> F-6P by enzyme phosphoglucose isomerase. This is a conversion of an aldose to a ketose. F-6P can be readily cleaved into two 3C fragments. * Isomerization of an aldose to a ketose; can be cleaved into 2, 3C units. STEP #3: F-6P -> F-1,6-BP by enzyme phosphofructokinase (PFK). PFK requires an input of ATP to function. * Second kinase reaction traps sugar in the fructose isomer. Energy is used in the 1st phase of glycolysis and recovered in the 2nd phase. Allosteric enzyme; PFK sets pace of glycolysis (regulation). STEP #4: F-1,6-BP -> GAP and DHAP by ezyme aldolase. GAP and DHAP are 2 3C fragments, and isomers of eachother. GAP will proceed through the remaining steps in glycolysis. * This is a readily reversible reaction, occurs near equilibrium. * From here on out, all reactions will consist of 3C units. STEP #5: Dihydroxyacetone phosphate -> Glyceraldehyde 3-phosphate by enzyme Triose Phosphate Isomerase. Enzyme TPI catalyzes the isomerization of these 3C phosphorylated sugars. * Rapid and reversible reaction, occurs near equilibrium. Equilibrium favors DHAP; however, remaining glycolysis steps utilize GAP thereby driving conversion DHAP -> GAP. STEP #6: GAP -> 1,3-BPG by enzyme Glyceraldehyde 3-phosphate dehydrogenase (GAP DH). This is a redox reaction. * Forms the acyl-phosphate 1,3-BPG (with high phosphoryl transfer potential). STEP #7: 1,3-BPG -> 3-Phosphoglycerate by enzyme Phosphoglycerate kinase. This enzyme catalyzes the transfer of a phosphoryl group from the acyl phosphate on 1,3-BPG to ADP. 1,3-BPG is the kinase substrate that has high phosphoryl transfer potential, and we get the products 3-phosphoglycerate and ATP. * Energy of previous oxidation trapped in 1,3-BPG. * 1,3-BPG has a high phosphoryl-transfer potential. * Example of substrate-level phosphorylation. * A kinase transfers phosphoryl group from 1,3-BPG to ADP to form ATP. STEP #8: 3-phosphoglycerate -> 2-phosphoglycerate by enzyme phosphoglycerate mutase. We shift the position of the phosphoryl group and we get the product 2-phosphoglycerate. * Rearrangement. * A mutase catalyzes an intramolecular shift of a chemical group. STEP #9: 2-phosphoglycerate -> phosphoenolpyruvate (PEP) by enzyme enolase. This is a dehydration reaction. A double bond gets introduced. * Introduces a double bond, creating enol phosphate. * PEP is an unstable molecule with greater phosphoryl-transfer potential than 2-PGA. STEP #10: Phosphoenolpyruvate -> pyruvate (enol form) -> pyruvate by enzyme pyruvate kinase. Pyruvate kinase transfers phosphoryl group to ADP generating ATP (our 2nd substrate level phosphorylation). * Remember: the 2nd stage of glycolysis occurs twice every one molecule of glucose, because we split that glucose into 2 3C fragments. - 2 ATP after step 7. - 2 ATP after step 10 (4 total). But we used 2 ATP in first stage so -> net: 2 atp per molecule of glucose during glycolysis. * Phosphoenolpyruvate has a high phosphoryl-transfer potential because phosphoryl group traps molecule in the unstable enol form. * 2nd substrate level phosphorylation reaction. * Removal of phosphoryl group allows conversion to the ketone form (pyruvate) which is more stable. * Reaction is virtually irreversible.

Which amino acids can be phosphorylated?

Serine, Threonine, Tyrosine (Only amino acids with a terminal -OH group)

Sketch and explain the signaling cascade initiated by EGF binding the EGF receptor. How might Ras be mutated in some cancer cells?

Some growth factors and hormones—such as epidermal growth factor (EGF), platelet-derived growth factor, and insulin—bind to the extracellular domains of transmembrane receptors that have tyrosine kinase domains within their intracellular domains. These receptor tyrosine kinases (RTKs) signal by mechanisms quite similar to those described for the pathway initiated by the growth-hormone receptor discussed in the preceding subsection. Humans have 58 known genes encoding receptor tyrosine kinases. Mutations in these receptors cause a range of pathologies, including arteriosclerosis, cancer, inflammation, and type 2 diabetes. Consider, for example, epidermal growth factor, a 6-kDa polypeptide that stimulates the growth of epidermal and epithelial cells by binding to the epidermal-growth-factor receptor, a single polypeptide chain consisting of an extracellular growth hormone binding domain, a helix that spans the membrane, and an intracellular kinase domain. The receptor is monomeric and enzymatically inactive in the absence of the growth factor. The binding of EGF to its extracellular domain causes the receptor to dimerize and undergo cross-phosphorylation and activation. How is the signal transferred beyond the receptor tyrosine kinase? A key adaptor protein, called Grb-2, links the phosphorylation of the EGF receptor to the stimulation of cell growth through a chain of protein phosphorylations (FIGURE 13.15). On phosphorylation of the receptor, Grb-2 binds to the phosphotyrosine residues of the receptor tyrosine kinase. Grb-2 then recruits a protein called Sos. Sos, in turn, binds to Ras and activates it. Ras is a very prominent signal-transduction component that we will consider shortly. Finally, Ras, in its activated form, binds to other components of the molecular circuitry, leading to the activation of the specific protein kinases that phosphorylate specific targets that promote cell growth. We see here another example of how a signal-transduction pathway is constructed. Specific protein-protein interactions link the original ligand-binding event to the final result—the stimulation of cell growth. The signal-transduction protein Ras is a member of a family of signal proteins—the small G proteins, or small GTPases. These small G proteins cycle between an active GTP-bound form and an inactive GDP-bound form. They differ from the heterotrimeric G proteins in being smaller (20-25 kDa compared with 40-45 kDa for Gα) and monomeric. Recall that Sos is the immediate upstream link to Ras in the circuit conveying the EGF signal (Figure 13.15). How does Sos activate Ras? Sos binds to Ras, reaches into the nucleotide-binding pocket, and opens it up, allowing GDP to escape and GTP to enter in its place. Sos is referred to as a guanine-nucleotide exchange factor (GEF). Like the Gα protein, Ras possesses an intrinsic GTPase activity, which serves to terminate the signal and return the system to the inactive state. This activity is slow but is augmented by helper proteins termed GTPase-activating proteins (GAPs). The guanine-nucleotide exchange factors and the GTPase-activating proteins allow the G-protein cycle to proceed with rates appropriate for a balanced level of downstream signaling. Ras might be mutated so that it loses its intrinsic GTPase activity, dissalowing it to terminate the signal and return the system to the inactive state. This could lead to uncontrolled cell growth. Genes that normally regulate cell growth often cause cancer when mutated. The gene encoding Ras, a component of the EGF-initiated pathway, is one of the genes most commonly mutated in human tumors. The most common mutation in tumors is a loss of the intrinsic GTPase activity. Thus, the Ras protein is trapped in the "on" position and continues to stimulate cell growth, even in the absence of a continuing signal.

Explain the two stages of glycolysis.

Stage 1: Utilizing ATP, ATP is required. - Glucose is trapped and cleaved intwo two interconvertile 3C molecules. These 3C units are then oxidized to pyruvate (later). Stage 2: ATP is generated. - Ultimate net gain of 2 ATP per molecule of glucose. Glycolysis can be thought of as comprising two stages (FIGURE 16.1). Stage 1 is the trapping and preparation phase. No ATP is generated in this stage. Stage 1 begins with the conversion of glucose into fructose 1,6-bisphosphate, which consists of three steps: a phosphorylation, an isomerization, and a second phosphorylation reaction. The strategy of these initial steps in glycolysis is to trap the glucose in the cell and form a compound that can be readily cleaved into phosphorylated three-carbon units. Stage 1 is completed with the cleavage of the fructose 1,6-bisphosphate into two phosphorylated three-carbon fragments. These resulting three-carbon units are readily interconvertible. In stage 2, ATP is harvested when the three-carbon fragments are oxidized to pyruvate.

What is substrate-level phosphorylation?

Substrate-level Phosphorylation: EX: The transfer of a phosphoryl group from the acyl phosphate on 1,3-BPG to ADP. The kinase substrate is 1,3-BPG and it has high phosphoryl transfer potential. EX: Phosphoenolpyruvate is catalyzed by pyruvate kinase to form pyruvate. Phosphoenolpyruvate has a high phosphoryl-transfer potential because the phosphoryl group traps the molecule in the unstable enol form. Pyruvate kinase transfers the phosphoryl from from PEP to ADP generating ATP. The enzyme-catalyzed formation of ATP by direct transfer of a phosphate group to ADP from an intermediate substrate in catabolism; ATP synthesis when the phosphate donor is a substrate with high phosphoryl transfer potential

How many molecules of ATP (net) and NADH are produced during glycolysis?

TOTAL ENERGY RELEASED * Glucose + 2 Pi + 2 ADP + 2 NAD+ -> 2 pyruvate + 2 ATP + 2 NADH + H+ + 2 H2O * Remember, the 2nd stage of glycolysis occurs twice every one molecule of glucose, because we split that glucose into 2 3C fragments. - 2 ATP after step 7. - 2 ATP after step 10 (now 4 total). - BUT, we used 2 ATP in the first stage so -> Net = 2 ATP per molecule of glucose during glycolysis.

Be able to fill in this table.

The "Downstream Signaling Pathway" section is only meant to be separated for the Beta-adrenergic receptor portion. Ignore it for the rest.

Understand the role of proteases in digestion.

The components of our meal must be degraded into small molecules for absorption by the epithelial cells of the intestine and for transport in the blood. Proteins are digested to amino acids by proteolytic enzymes (proteases) secreted by the stomach and pancreas. Polysaccharides such as starchare cleaved into monosaccharides by α-amylase from the pancrease and to a lesser extent in saliva. Lipids are converted into fatty acids by lipases secreted by the pancreas. All of the digestive enzymes are hydrolases; that is, they cleave their substrates by the addition of a molecule of water. Digestion prepares large biomolecules for use in metabolism. Proteases digest proteins into amino acids and peptides. Protein digestion starts in the stomach and continues in the intestine.

How do G proteins become active and inactive? Review similarities and differences between Gs and Gq, and Ras.

The conformational change in the cytoplasmic domain of the receptor activates a GTP-binding protein. This signal-coupling protein is called a G protein. In the unactivated state, the guanyl nucleotide bound to the G protein is GDP. In this form, the G protein exists as a heterotrimer consisting of α, β, γ subunits; the α subunit (referred to as Gα) binds the nucleotide. The α and γ subunits are usually anchored to the membrane covalently attached to fatty acids. The exchange of the bound GDP for GTP is catalyzed by the ligand-bound receptor. The ligand-receptor complex interacts with the heterotrimeric G protein and opens the nucleotide-binding site so that GDP can depart and GTP can bind. The α subunit simultaneously dissociates from the βγ dimer. This dissociation transmits the signal that the receptor has bound to the ligand. The Gα protein binds to adenylate cyclase on the Gα surface that had bound the βγ dimer when the Gα protein was in its GDP form. Gαs ("s" stands for stimulatory) stimulates adenylate cyclase activity, thus increasing cAMP production. Using a different pathway, phosphoinositide cascade, like the adenylate cyclase cascade, converts extracellular signals into intracellular ones. The intracellular messengers formed by activation of the pathway arise from the cleavage of PIP2, a membrane phospholipid. The binding of a hormone such as vasopressin to its 7TM receptor leads to the activation of PLC. The Gα protein that activates PLC is called Gαq. Ras is a signal transduction protein. Sos is the immediate upstream link to Ras in the circuit conveying the EGF signal. Sos binds to Ras, reaches into the nucleotide-binding pocket, and opens it up, allowing GDP to escape and GTP to enter in its place. Ras possesses an intrinsic GTPase activity, which serves to terminate the signal and return the system to the inactive state. Gαq is hetermotrimeric, just like Gαs. All of these are G-Proteins, so they all have to have GTP bound to them in order to be active, and they all have GTP intrinsic activity. Ras on the other hand, still has GTP intrinsic activity, but is monomeric.

Understand the purpose and function of the Cori Cycle.

The process of using lactate produced in muscles through anaerobic respiration heading into the blood stream and then being taken up by the liver to produce glucose: the Cori Cycle. - Metabolic cooperation between muscle and liver, it removes lactate from the blood. Lactate is not only removed, but it is also then used to generate something very valuable, which is glucose. Lactate produced by active skeletal muscle and red blood cells is a source of energy for other organs. Red blood cells lack mitochondria and can never oxidize glucose completely. Recall that in contracting type IIb skeletal muscle during vigorous exercise, the rate at which glycolysis produces pyruvate exceeds the rate at which the citric acid cycle oxidizes it. In these cells, lactate dehydrogenase reduces excess pyruvate to lactate to restore redox balance (p. 321). However, lactate is a dead end in metabolism. It must be converted back into pyruvate before it can be metabolized. Lactate and protons are transported out of these cells into the blood. In contracting skeletal muscle, the formation and release of lactate lets the muscle generate ATP in the absence of oxygen and shifts the burden of metabolizing lactate from muscle to other organs. The lactate in the bloodstream has two fates. In one fate, the plasma membranes of some cells—particularly cells in cardiac muscle and slow-twitch (type 1) skeletal muscle—contain carriers that make the cells highly permeable to lactate. The molecule diffuses from the blood into such permeable cells. Inside these well-oxygenated cells, lactate can be reverted back to pyruvate and metabolized through the citric acid cycle and oxidative phosphorylation to generate ATP. The use of lactate in place of glucose by these cells makes more circulating glucose available to the active muscle cells. In the other fate, excess lactate enters the liver and is converted first into pyruvate and then into glucose by the gluconeogenic pathway. Thus, the liver restores the level of glucose necessary for active muscle cells, which derive ATP from the glycolytic conversion of glucose into lactate. These reactions constitute the Cori cycle (FIGURE 17.11). * FIGURE 17.11 The Cori cycle. Lactate formed by active muscle is converted into glucose by the liver. This cycle shifts part of the metabolic burden of active muscle to the liver. The symbol ~P represents nucleoside triphosphates.

What is the purpose and definition of gluconeogensis?

The purpose of gluconeogenesis is to start with pyruvate and end with glucose. * The synthesis of glucose from noncarbohydrate precursors, a process called gluconeogenesis. * Gluconeogenesis in the liver and kidney helps to maintain the glucose concentration in the blood, from which it can be extracted by the brain and muscle to meet their metabolic demands. Gluconeogenesis is relevant for periods of fasting > 1 day (pyruvate -> glucose). * It is the conversion of noncarbohydrate precursors to glucose: lactate, amino acids, and glycerol. These three precursors can all be converted into pyruvate. We are producing glucose from precursors. * The pathway is not a complete reversal of glycolysis. Some steps are energetically unfavorable (glycolysis reactions with large -ΔG value) and must be catalyzed by their own separate enzyme (different from glycolysis) with a different ΔG value. - Bypass steps in gluconeogenesis correspond to steps 1, 3, and 10 in glycolysis. Actual ΔG°' for 2 pyruvate -> 1 glucose - -38 kJ/mol. - Requires 4 ATP, 2 GTP, and 2 NADH. * Remember Gluconeogenesis isn't a reverse of glycolysis because the equilibrium of glycolysis lies far on the side of pyruvate formation. * These reactions are in equilibrium, so when gluconeogenesis is favored, the reverse reactions will take place until the next irreversible step is reached (pink steps). The last enzyme (glucose 6-phosphate) is only in the liver because the liver helps to maintain blood glucose levels so that the glucose can be extracted by the brain and muscle to meet metabolic demands. We don't want other tissues to use up that glucose just because it's available. so keep G-6P trapped in the cell unless you're in liver tissue and then go ahead and proceed to the production of glucose.

Fuels are oxidized in cells to provide energy. Why does oxidation of fats provide more energy than oxidation of carbs?

We're looking at the free energy of oxidation of some common single carbon compounds. Fuels are oxidized one carbon at a time, and the more reduced a carbon is to begin with, the more free energy will be released. Why? When electrons move from an atom with low affinity for an electron like carbon here, to an atom with high affinity, for an electron like oxygen, energy will be released. Methane has the most energy, most reduced. Carbon dioxide has the least energy. It's the most oxidized. Fatty acids can provide more energy than carbs because the carbon in fats is more reduced. The carbons in fatty acids have more electrons around them compared to the carbons in glucose. A greater number of electrons around these carbon atoms in fatty acids will be transferred to oxygen as fatty acids are oxidized and more energy will be released than when those same processes happen to carbs. Carbon atoms in fatty acids have more e- around them. When e- move from an atom with low affinity for e- (low electronegatvitity, like C) to an atom with high affinity for e- (like O), energy is released. So, the greater the number of e- around the carbon atoms in fatty acids are transferred to O2 (fatty acids are oxidized), more energy is released than when the same process happens to carbs.