MTC Module 2: Glycolysis

What determines the importance of glycolysis in our fuel economy?

The importance of glycolysis in our fuel economy is related to the availability of glucose in the blood, as well as the ability of glycolysis to generate ATP in both the presence and absence of O2.

What are the key regulatory enzymes of glycolysis?

The key regulated enzymes of glycolysis are hexokinase, phosphofrutokinase-1, and pyruvate kinase.

Explain the role of the liver in biosynthesis

The liver is the major site of biosynthetic reactions in the body. In addition to those pathways mentioned previously, the liver synthesizes fatty acids from the pyruvate generated by glycolysis. It also synthesizes glucose from lactate, glycerol 3-phosphate, and amino acids in the gluconeogenic pathway, which is principally a reversal of glycolysis. Consequently, in liver, many of the glycolytic enzymes exist as isoenzymes with properties suited for these functions.

Give a summary of the glycolytic pathway and its delta G.

The overall net reaction in the glycolytic pathway is: Glucose + 2NAD+ + 2Pi + 2ADP --> 2Pyruvate + 2NADH + 4H+ + 2ATP + 2H2O -The pathway occurs with an overall negative DG0' of approximately -22 kcal. Therefore, it cannot be reversed without the expenditure of energy.

Explain how glycolysis generates ATP.

The oxidation of glucose to pyruvate generates ATP from substrate-level phosphorylation (the transfer of phosphate from high-energy intermediates of the pathway to ADP) and NADH. Subsequently, the pyruvate may be oxidized to CO2 in the TCA cycle and ATP generated from electron transfer to oxygen in oxidative phosphorylation. However, if the pyruvate and NADH from glycolysis are converted to lactate (anaerobic glycolysis), ATP can be generated in the absence of oxygen, via substrate-level phosphorylation.

What happens to the pyruvate generated during glycolysis?

The pyruvate generated during glycolysis can enter the mitochondria and be oxidized completely to CO2 by pyruvate dehydrogenase and the TCA cycle.

What are the phases of glycolysis?

The glycolytic pathway, which cleaves 1 mole of glucose to 2 moles of the 3-carbon compound pyruvate, consists of a preparative phase and an ATP-generating phase.

Lactate and pyruvate are in equilibrium in the cell, and the ratio of lactate to pyruvate reflects the NADH/NAD+ ratio. Both acids are released into blood, and the normal ratio of lactate to pyruvate in blood is approximately 25:1. This ratio can provide a useful clinical diagnostic tool. Because lactic acidemia can be the result of a number of problems, such as hypoxia, MERFF, thiamine deficiency, and pyruvate dehdyrogenase deficiency, under which of these conditions would you expect the lactate/pyruvate ratio in blood to be much greater than normal?

Hypoxia and inherited deficiencies of subunits in the electron transport chain impair NADH oxidation, resulting in a higher NADH/NAD+ ratio in the cell, and, therefore, a higher lactate/pyruvate ratio in blood. In contrast, conditions that cause lactic acidemia as a result of defects in the enzymes of pyruvate metabolism (thiamine deficiency or pyruvate dehy- drogenase deficiency) would increase both pyruvate and lactate in the blood and have little effect on the ratio.

Explain the allosteric regulation of PFK-1 by AMP and ATP

-ATP binds to two different sites on the enzyme, the substrate binding site and an allosteric inhibitory site. Under physiologic conditions in the cell, the ATP concentration is usually high enough to saturate the substrate binding site and inhibit the enzyme by binding to the ATP allosteric site. -This effect of ATP is opposed by AMP, which binds to a separate allosteric activator site (Figure 22.14). For most of the PFK-1 isoenzymes, the binding of AMP increases the affinity of the enzyme for fructose 6-P (e.g., shifts the kinetic curve to the left). -Thus, increases in AMP concentration can greatly increase the rate of the enzyme (see Fig. 22.14), particularly when fructose-6-P concentrations are low.

Explain the allosteric regulation of PFK-1 by Fructose-2,6-BisP

-Fructose-2,6-bisP is also an allosteric activator of PFK-1 that opposes the ATP inhi- bition. Its effect on the rate of activity of PFK-1 is qualitatively similar to that of AMP, but it has a separate binding site. -Fructose-2,6-bisP is NOT an intermediate of glycolysis but is synthesized by an enzyme that phosphorylates fructose 6-phosphate at the 2 position. The enzyme is therefore named phosphofructokinase-2 (PFK-2); it is a bifunctional enzyme with two separate domains, a kinase domain and a phosphatase domain. At the kinase domain, fructose-6-P is phosphorylated to fructose-2,6-bisP and at the phosphatase domain, fructose-2,6-bisP is hydrolyzed back to fructose-6-P. -PFK-2 is regulated through changes in the ratio of activity of the two domains. For example, in skeletal muscles, high concentrations of fructose- 6-P activate the kinase and inhibit the phosphatase, thereby increasing the concentration of fructose-2,6-bisP and activating glycolysis.

What is hexokinase called in the liver and in pancreatic B-Cells?

-Glucokinase -Has a much higher Km

What are the fates of the products of glycolysis?

-Glycolysis occurs in the cytosol and generates cytosolic NADH. Because NADH cannot cross the inner mitochondrial membrane, its reducing equivalents are transferred to the electron transport chain by either the malate-aspartate shuttle or the glycerol 3-phopshate shuttle (see Fig. 22.1). -Pyruvate is then oxidized completely to CO2 by pyruvate dehydrogenase and the TCA cycle. Complete aerobic oxidation of glucose to CO2 can generate approximately 30 to 32 moles of ATP per mole of glucose.

Describe the Regulation of Hexokinases

-Hexokinases exist as tissue-specific isoenzymes whose regulatory properties reflect the role of glycolysis in different tissues. In most tissues, hexokinase is a low-Km enzyme with a high affinity for glucose (see Chapter 9). -It is inhibited by physiologic concentrations of its product, glucose-6-P (see Fig. 22.12). If glucose-6-P does not enter glycolysis or another pathway, it accumulates and decreases the activity of hexokinase. -In the liver, the isoenzyme glucokinase is a high-Km enzyme that is not readily inhibited by glucose-6-P. Thus, glycolysis can continue in liver even when energy levels are high so that anabolic pathways, such as the synthesis of the major energy storage compounds, glycogen and fatty acids, can occur.

How is glycolysis regulated?

-In each cell, glycolysis is regulated to ensure that ATP homeostasis is maintained, without using more glucose than necessary. -In most cell types, hexokinase (HK), the first enzyme of glycolysis, is inhibited by glucose 6-phosphate (see Fig. 22.1). Thus, glucose is not taken up and phosphorylated by a cell unless glucose-6-P enters a metabolic pathway, such as glycolysis or glycogen synthesis. -The control of glucose-6-P entry into glycolysis occurs at phosphofructokinase-1 (PFK-1), the rate-limiting enzyme of the pathway, PFK-1 is allosterically inhibited by ATP and allosterically activated by AMP. AMP increases in the cytosol as ATP is hydrolyzed by energy-requiring reactions.

Explain how other tissues use lactate as energy

-In many other tissues, lactate is oxidized to pyruvate, which is then oxidized to CO2 in the TCA cycle. Although the equilibrium of the lactate dehydrogenase reaction favors lactate production, flux occurs in the opposite direction if NADH is being rap- idly oxidized in the electron transport chain (or being used for gluconeogenesis) -The heart, with its huge mitochondrial content and oxidative capacity, is able to use lactate released from other tissues as a fuel. During an exercise such as bicycle riding, lactate released into the blood from skeletal muscles in the leg might be used by resting skeletal muscles in the arm. In the brain, glial cells and astrocytes pro- duce lactate, which is used by neurons or released into the blood.

What is lactate dehydrogenase (LDH)?

-Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. It converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver. At high concentrations of lactate, the enzyme exhibits feedback inhibition, and the rate of conversion of pyruvate to lactate is decreased.

What is lactic acidosis?

-Lactate levels > 5 mM; blood pH < 7.2. -Generally results from increased NADH/NAD+ ratio in tissues e.g. consumption of large amounts of alcohol......

What is the cause of lactic acidosis?

-Lactic acidosis generally results from a greatly increased NADH/NAD+ ratio in tissues (Fig.22.15). The increased NADH concentration prevents pyruvate oxida- tion in the TCA cycle and directs pyruvate to lactate. To compensate for the decreased ATP production from oxidative metabolism, PFK-1, and, therefore, the entire glycolytic pathway is activated. For example, consumption of high amounts of alcohol, which is rapidly oxidized in the liver and increases NADH levels, can result in a lactic acidosis. Hypoxia in any tissue increases lactate production as cells attempt to compensate for a lack of O2 for oxidative phosphorylation. A number of other problems that interfere either with the electron transport chain or pyruvate oxidation in the TCA cycle result in lactic acidemia (see Fig.22.15). For example, OXPHOS diseases (inherited deficiencies in subunits of complexes in the electron transport chain, such as MERFF) increase the NADH/NAD+ ratio and inhibit PDH (see Chapter 21). Impaired PDH activity from an inherited deficiency of E1 (the decarboxylase subunit of the complex), or from severe thiamine defi- ciency, increases blood lactate levels (see Chapter 20). Pyruvate carboxylase defi- ciency also can result in lactic acidosis (see Chapter 20), because of an accumula- tion of pyruvate. -Lactic acidosis can also result from inhibition of lactate utilization in gluconeogenesis (e.g., hereditary fructose intolerance, which is due to a defective aldolase gene). If other pathways that use glucose-6-P are blocked, glucose-6-P can be shunted into glycolysis and lactate production (e.g., glucose 6-phosphatase deficiency).

Explain the mechanism of the malate-aspartate shuttle.

-Many tissues contain both the glycerol-3-P shuttle and the malate-aspartate shuttle. -In the malate-aspartate shuttle (Fig. 22.8), cytosolic NAD+ is regenerated by cytosolic malate dehydrogenase, which transfers electrons from NADH to cytosolic oxaloacetate to form malate. Malate is transported across the inner mitochondrial membrane by a specific translocase, which exchanges malate for Alpha-ketoglutarate. In the matrix, malate is oxidized back to oxaloacetate by mitochondrial malate dehydrogenase, and NADH is generated. This NADH can donate electrons to the electron transport chain with generation of approximately 2.5 moles of ATP per mole of NADH. -The newly formed oxaloacetate cannot pass back through the inner mitochondrial membrane under physiologic conditions, so aspartate is used to return the oxaloacetate carbon skeleton to the cytosol. In the matrix, transamination reactions transfer an amino group to oxaloacetate to form aspartate, which is trans- ported out to the cytosol (using an aspartate/glutamate exchange translocase) and converted back to oxaloacetate through another transamination reaction.

Explain the regulation of PFK-2

-PFK-2 is regulated through changes in the ratio of activity of the two domains. For example, in skeletal muscles, high concentrations of fructose- 6-P activate the kinase and inhibit the phosphatase, thereby increasing the concentration of fructose-2,6-bisP and activating glycolysis. -PFK-2 also can be regulated through phosphorylation by serine/threonine protein kinases. The liver isoenzyme contains a phosphorylation site near the amino terminal that decreases the activity of the kinase and increases the phosphatase activity. This site is phosphorylated by the cAMP-dependent protein kinase (protein kinase A) and is responsible for decreased levels of liver fructose-2,6-bisP during fasting conditions (as modulated by circulating glucagon levels, which is discussed in detail in Chapters 26 and 31). -The cardiac isoenzyme contains a phosphorylation site near the carboxy terminal that can be phosphorylated in response to adrenergic activators of contraction (such as norepinephrine) and by increased AMP levels. Phosphorylation at this site increases the kinase activity and increases fructose-2, 6-bisP levels, thereby contributing to the activation of glycolysis.

Describe the 2,3-BPG shunt.

-The 1,3-BPG that is produced during glycolysis can be transformed into 2,3-BPG and used as an allosteric inhibitor of hemoglobin. 1. 1,3-BPG acted on by mutase 2. Forms 2,3-BPG 3. 2,3-BPG can be acted on by a phosphatase, transform into 3PG, and be used in glycolysis

Explain the Relationship between ATP, ADP, and AMP Concentrations

-The AMP levels within the cytosol provide a better indicator of the rate of ATP utilization than the ATP concentration itself (Fig. 22.13). -The concentration of AMP in the cytosol is determined by the equilibrium position of the adenylate kinase reaction. The equilibrium is such that hydrolysis of ATP to ADP in energy-requiring reactions increases both the ADP and AMP contents of the cytosol. However, ATP is present in much higher quantities than AMP or ADP, so that a small decrease of ATP concentration in the cytosol causes a much larger percentage increase in the small AMP pool. -In skeletal muscles, for instance, ATP levels are approximately 5 mM and decrease by no more than 20% during strenuous exercise (see Fig. 22.13). At the same time, ADP levels may increase by 50%, and AMP levels, which are in the micromolar range, increase by 300%. -AMP activates a number of metabolic pathways, including glycolysis, glycogenolysis, and fatty acid oxidation (particu- larly in muscle tissues), to ensure that ATP homeostasis is maintained.

What determines the fate of pyruvate?

-The fate of pyruvate depends on the route used for NADH oxidation. If NADH is reoxidized in a shuttle system, pyruvate can be used for other pathways, one of which is oxidation to acetyl-CoA and entry into the TCA cycle for complete oxidation. -Alternatively, in anaerobic glycolysis, pyruvate is reduced to lactate and diverted away from other potential pathways. Thus, the use of the shuttle systems allows for more ATP to be generated than by anaerobic glycolysis by both oxidiz- ing the cytoplasmically derived NADH in the electron transport chain and by allow- ing pyruvate to be oxidized completely to CO2.

Explain the mechanism that is exploited by NADH mitochondrial shuttle systems in order to oxidize NADH and transport reducing equivalents to the ETC.

-The inner mitochondrial membrane is impermeable to NADH. Consequently, NADH is reoxidized to NAD+ IN THE CYTOSOL by a reaction that transfers the electrons to DHAP in the glycerol 3-phosphate (glycerol- 3-P) shuttle and oxaloacetate in the malate-aspartate shuttle. -The NAD+ that is formed in the cytosol returns to glycolysis while glycerol-3-P or malate carry the reducing equivalents that are ultimately transferred across the inner mitochondrial membrane. -Thus, these shuttles transfer electrons and not NADH per se.

What is the first committed step of glycolysis?

-The next step of glycolysis, phosphorylation of fructose-6-P to fructose 1,6- bisphosphate (fructose-1,6-bisP) by phosphofructokinase-1 (PFK-1), is generally considered the first committed step of the pathway. This phosphorylation requires ATP and is thermodynamically and kinetically irreversible. Therefore, PFK-1 irrevocably commits glucose to the glycolytic pathway. -PFK-1 is a regulated enzyme in cells, and its regulation controls the entry of glucose into glycolysis. Like hexokinase, it exists as tissue-specific isoenzymes whose regulatory properties match variations in the role of glycolysis in different tissues.

What do cells do when they have a limited supply of oxygen?

-When cells have a limited supply of oxygen (e.g., kidney medulla), or few or no mitochondria (e.g., the red cell), or greatly increased demands for ATP (e.g., skeletal muscle during high- intensity exercise), they rely on anaerobic glycolysis for generation of ATP. In anaerobic glycolysis, lactate dehydrogenase oxidizes the NADH generated from glycolysis by reducing pyruvate to lactate (Fig. 22.2). -Because O2 is not required to reoxidize the NADH, the pathway is referred to as anaerobic. The energy yield from anaerobic glycolysis (2 moles of ATP per mole of glucose) is much lower than the yield from aerobic oxidation -The lactate (lactic acid) is released into the blood. Under pathologic condition that cause hypoxia, tissues may generate enough lactic acid to cause lactic acidemia.

Describe the action of glyceraldehyde-3-phosphate dehydrogenase.

1. GPDH oxidizes the aldehyde group of G- 3-P to a carboxyl group and transfers the electrons to NAD+ to form NADH. 2. The cysteine residue in GPDH forms a high-energy thioester bond and accepts an inorganic phosphate to form a high- energy acyl phosphate bond. This is called substrate-level phosphorylation.

Given one molecule of glucose, how much ATP is produced?

1. Glycolysis: 2 ATP 2. Oxidative Phosphorylation: 25 ATP 3. G3P Shuttle: 3 ATP -OR- 3. Malate-Aspartate shuttle: 5 ATP

What is the fate of the NADH produced during glycoysis and its impact on the fate of pyruvate?

1. NADH must be continuously reoxidized back to NAD+ to provide an electron acceptor for GPDH so that glycolysis can continue. 2. The fate of pyruvate depends on the route used for NADH oxidation

Explain the tissue-specific nature of the regulatory enzymes of glycolysis

All of the regulatory enzymes of glycolysis exist as tissue-specific isoenzymes, which alter the regulation of the pathway to match variations in conditions and needs in different tissues. For example, in the liver, an isoenzyme of pyruvate kinase introduces an additional regulatory site in glycolysis that contributes to the inhibi- tion of glycolysis when the reverse pathway, gluconeogenesis, is activated.

What is the advantage of anaerobic respiration?

Anaerobic glycolysis generates energy in cells with a limited supply of oxygen or few mitochondria.

Explain the acid production of anaerobic respiration.

Anaerobic glycolysis results in acid production in the form of H. Glycolysis forms pyruvic acid, which is reduced to lactic acid. At an intracellular pH of 7.35, lactic acid dissociates to form the carboxylate anion, lactate, and H+ (the pKa for lactic acid is 3.85). Lactate and the H+ are both transported out of the cell into interstitial fluid by a transporter on the plasma membrane and eventu- ally diffuse into the blood. If the amount of lactate generated exceeds the buffering capacity of the blood, the pH drops below the normal range, resulting in lacticacidosis (see Chapter 4).

Why is Glucose-6-Phosphate A branch point in carbohydrate metabolism?

At this point, G6P can either: 1. Continue down glycolysis 2. Go to the pentose phosphate pathway 3. Go to glycogen synthesis 4. Contribute to other pathways

4. Which of the following statements correctly describes an aspect of glycolysis? (A) ATP is formed by oxidative phosphorylation. (B) 2 ATP are used in the beginning of the pathway. (C) Pyruvate kinase is the rate-limiting enzyme. (D) One pyruvate and three CO2 are formed from the oxidation of one glucose molecule. (E) The reactions take place in the matrix of the mitochondria.

B

A major role of glycolysis is which of the following? (A) To synthesize glucose (B) To generate energy (C) To produce FAD(2H) (D) To synthesize glycogen (E) To use ATP to generate heat

B

Describe pyruvate dehydrogenase,

Can be Inactivated through rapid phosphorylation. Activators: ADP, Ca2+ Inhibitor: NADH, acetyl CoA

2. Starting with glyceraldehyde 3-phosphate and synthesizing one molecule of pyruvate, the net yield of ATP and NADH would be which of the following? (A) 1 ATP, 1 NADH (B) 1 ATP, 2 NADH (C) 1 ATP, 4 NADH (D) 2 ATP, 1 NADH (E) 2 ATP, 2 NADH (F) 2 ATP, 4 NADH (G) 3 ATP, 1 NADH (H) 3 ATP, 2 NADH (I) 3 ATP, 4 NADH

D

5. How many moles of ATP are generated by the complete aerobic oxidation of 1 mole of glucose to 6 moles of CO2? (A) 2-4 (B) 10-12 (C) 18-22 (D) 30-32 (E) 60-64

D

What cells can use glycolysis?

Every cell type in the human is able to generate adenosine triphosphate (ATP) from glycolysis, the pathway in which glucose is oxidized and cleaved to form pyruvate .

What happens to fructose-1,6-bisP?

Fructose-1,6-bisP is cleaved into two phosphorylated 3-carbon compounds (triose phosphates) by aldolase (see Fig. 22.5). Dihydroxyacetone phosphate (DHAP) is isomerized to glyceraldehyde 3-phosphate (glyceraldehyde-3-P), which is a triose phosphate. Thus, for every mole of glucose entering glycolysis, 2 moles of glyceraldehyde-3-P continue through the pathway.

What biosynthetic pathway does the conversion of G6P to 1,3-BPG and Acetyl CoA contribute to?

G6P to 1,3-BPG: Glycerol-P Acetyl Coa: Fatty Acids They combine to form triglycerides.

What is the overall reaction of glycolysis?

Glucose + 2 NAD+ +2 Pi +2 ADP--> 2 Pyruvate+2 NADH+2 H+ +2 ATP + 2 H2O

How important is glucose to energy metabolism?

Glucose is the major sugar in our diet; all cells can utilize glucose for energy.

What is the first enzymatic step of glycolysis?

Glucose metabolism begins with transfer of a phosphate from ATP to glucose to form glucose-6-P (Fig. 22.4). Phosphorylation of glucose commits it to metabolism within the cell because glucose-6-P cannot be transported back across the plasma membrane. The phosphorylation reaction is irreversible under physiologic conditions because the reaction has a high negative DG0'. Phosphorylation does not, however, commit glucose to glycolysis.

How is G6P a branch point in carbohydrate metabolism?

Glucose-6-P is a branchpoint in carbohydrate metabolism. It is a precursor for almost every pathway that uses glucose, including glycolysis, the pentose phosphate pathway, and glycogen synthesis. From the opposite point of view, it also can be generated from other pathways of carbohydrate metabolism, such as glycogenoly- sis (breakdown of glycogen), the pentose phosphate pathway, and gluconeogenesis (the synthesis of glucose from non-carbohydrate sources).

How does glycolysis start, and what does it yield?

Glycolysis begins with the phosphorylation of glucose to glucose 6-phosphate (glucose- 6-P) by hexokinase (HK). In subsequent steps of the pathway, one glucose-6-P molecule is oxidized to two pyruvate molecules with generation of two molecules of NADH (Fig. 22.1). A net generation of two molecules of ATP occurs through direct transfer of high-energy phosphate from intermediates of the pathway to ADP (substrate level phosphorylation).

What is generated by glycolysis?

Glycolysis generates 2 molecules of ATP through substrate-levels phosphorylation, and 2 molecules of NADH.

What does glycolysis do besides generate ATP?

Glycolysis has functions in addition to ATP production. For example, in liver and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosynthesis. Glycolysis also provides precursors for the synthesis of compounds such as amino acids and 5-carbon sugar phosphates.

What happens in the pathway of glycolysis?

Glycolysis is the pathway in which glucose is oxidized and cleaved to form pyruvate.

What molecules can be generated from intermediates in glycolysis?

Glycolysis, in addition to providing ATP, generates precursors for biosynthetic path- ways (Fig. 22.11). Intermediates of the pathway can be converted to ribose 5- phosphate, the sugar incorporated into nucleotides such as ATP. Other sugars, such as UDP-glucose, mannose, and sialic acid, are also formed from intermediates of glycolysis. Serine is synthesized from 3-phosphoglycerate, and alanine from pyruvate. The backbone of triacylglycerols, glycerol 3-phosphate, is derived from dihydroxyacetone phosphate in the glycolytic pathway.

3. When glycogen is degraded, glucose 1-phosphate is formed. Glucose 1-phosphate can then be isomerized to glucose 6-phosphate. Starting with glucose 1-phosphate, and ending with 2 molecules of pyruvate, what is the net yield of glycolysis, in terms of ATP and NADH formed? (A) 1 ATP, 1 NADH (B) 1 ATP, 2 NADH (C) 1 ATP, 3 NADH (D) 2 ATP, 1 NADH (E) 2 ATP, 2 NADH (F) 2 ATP, 3 NADH (G) 3 ATP, 1 NADH (H) 3 ATP, 2 NADH (I) 3 ATP, 3 NADH

H

What are hexokinases?

Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a family of tissue-specific isoenzymes that differ in their kinetic properties. The isoenzyme found in liver and Beta cells of the pancreas has a much higher Km than other hexokinases and is called glucokinase. In many cells, some of the hexokinase is bound to porins in the outer mitochondrial membrane (voltage-dependent anion channels; see Chapter 21), which gives these enzymes first access to newly synthesized ATP as it exits the mitochondria.

Other than a pathway of energy transformation, what is glycolysis?

In addition to serving as an anaerobic and aerobic source of ATP, glycolysis is an anabolic pathway that provides biosynthetic precursors. For example, in liver and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosyn- thesis. Glycolysis also provides precursors for the synthesis of compounds such as amino acids and ribose-5-phosphate, the precursor of nucleotides.

What happens in the ATP generating phase of glycolysis?

In the ATP-generating phase, glyceraldehyde 3-phosphate (a triose phosphate) is oxidized by NAD+ and phosphorylated using inorganic phosphate. The high- energy phosphate bond generated in this step is transferred to ADP to form ATP. The remaining phosphate is also rearranged to form another high-energy phosphate bond that is transferred to ADP. Because there were 2 moles of triose phosphate formed, the yield from the ATP-generating phase is 4 ATP and 2 NADH. The result is a net yield of 2 moles of ATP, 2 moles of NADH, and 2 moles of pyruvate per mole of glucose.

What are the energy-generating steps as pyruvate is completely oxidized to carbon dioxide to generate 12.5 molecules of ATP per pyruvate?

In the complete oxidation of pyru- vate to carbon dioxide, four steps generate NADH (pyruvate dehydrogenase, isocitrate dehydrogenase, A-ketoglutarate dehydrogenase, and malate dehydro- genase). One step generates FAD(2H) (succinate dehydrogenase), and one substrate level phosphorylation (succinate thiokinase). Thus, because each NADH generates 2.5 ATPs, the overall contribution by NADH is 10 ATP molecules. The FAD(2H) generates an additional 1.5 ATP, and the substrate-level phosphorylation provides one more. There- fore, 10 + 1.5 + 1 = 12.5 molecules of ATP.

What happens in the preparative phase of glycolysis?

In the initial preparative phase of glycolysis, glucose is phosphorylated twice by ATP and cleaved into two triose phosphates. The ATP expenditure in the beginning of the preparative phase is sometimes called "priming the pump," because this initial utilization of 2 moles of ATP/ mole of glucose results in the production of 4 moles of ATP/mole of glucose in the ATP-generating phase.

What is different about hexokinase regulation in the liver (called glucokinase)?

In the liver, glucokinase is not readily inhibited by G-6-P.

What happens to glyceraldehyde 3 phosphate?

In the next part of the glycolytic pathway, glyceraldehyde-3-P is oxidized and phosphorylated so that subsequent intermediates of glycolysis can donate phosphate to ADP to generate ATP. The first reaction in this sequence, catalyzed by glyceralde- hyde-3-P dehydrogenase, is really the key to the pathway (see Fig. 22.5). This enzyme oxidizes the aldehyde group of glyceraldehyde-3-P to an enzyme-bound carboxyl group and transfers the electrons to NAD+ to form NADH. The oxidation step is dependent on a cysteine residue at the active site of the enzyme, which forms a high-energy thioester bond during the course of the reaction. The high-energy intermediate immediately accepts an inorganic phosphate to form the high-energy acyl phosphate bond in 1,3-bisphosphoglycerate, releasing the product from the cysteine residue on the enzyme. This high-energy phosphate bond is the start of substrate-level phosphorylation (the formation of a high-energy phosphate bond where none previously existed, without the utilization of oxygen).

What happens to 1,3-BPG?

In the next reaction, the phosphate in this bond is transferred to ADP to form ATP by 3-phosphoglycerate kinase. The energy of the acyl phosphate bond is high enough (~13 kcal/mole) so that transfer to ADP is an energetically favorable process. 3-phosphoglycerate is also a product of this reaction.

Briefly summarize the steps that transform G6P into two 3-carbon fragments.

In the remainder of the preparative phase of glycolysis, glucose-6-P is isomerized to fructose 6-phosphate (fructose-6-P), again phosphorylated, and subsequently cleaved into two 3-carbon fragments (Fig 22.5). The isomerization, which positions a keto group next to carbon 3, is essential for the subsequent cleavage of the bond between carbons 3 and 4.

Detail the balance of anaerobic and aerobic respiration in tissues that possess a medium number of mitochondria.

In tissues with some mitochondria, both aerobic and anaerobic glycolysis occur simultaneously. The relative proportion of the two pathways depends on the mito- chondrial oxidative capacity of the tissue and its oxygen supply and may vary between cell types within the same tissue because of cell distance from the capil- laries. When a cell's energy demand exceeds the capacity of the rate of the electron transport chain and oxidative phosphorylation to produce ATP, glycolysis is acti- vated, and the increased NADH/NAD+ ratio will direct excess pyruvate into lactate. Because under these conditions pyruvate dehydrogenase, the TCA cycle, and the electron transport chain are operating as fast as they can, anaerobic glycolysis is meeting the need for additional ATP.

Describe isozymes of pyruvate kinase.

Isoenzymes: 1. R (red blood cells) 2. L (liver) -Can be inhibited through phosphorylation by cAMP and PKA during fasting. 3. M1/M2 (muscle and other tissues) -Activators: fructose 1,6-bis-P -Inhibitor: ATP

Explain the fate of lactate

Lactate released from cells undergoing anaerobic glycolysis is taken up by other tis- sues (primarily the liver, heart, and skeletal muscle) and oxidized back to pyruvate. In the liver, the pyruvate is used to synthesize glucose (gluconeogenesis), which is returned to the blood. The cycling of lactate and glucose between peripheral tissues and liver is called the Cori cycle (Fig. 22.10).

What happens if there are mutations in the TCA Cycle or ETC that prevents the products of glycolysis from moving on in metabolism?

Mutations in the TCA cycle, or proteins involved in oxidative phosphorylation, can lead to accumulation of pyruvate, which will lead to LACTIC ACIDEMIA.

Generalize the role of ATP in regulation of glycolysis

One of the major functions of glycolysis is the generation of ATP, and, therefore, the pathway is regulated to maintain ATP homeostasis in all cells. Phosphofruc- tokinase-1 (PFK-1) and pyruvate dehydrogenase (PDH), which links glycolysis and the TCA cycle, are both major regulatory sites that respond to feedback indi- cators of the rate of ATP utilization (Fig. 22.12). The supply of glucose-6-P for glycolysis is tissue dependent and can be regulated at the steps of glucose trans- port into cells, glycogenolysis (the degradation of glycogen to form glucose), or the rate of glucose phosphorylation by hexokinase isoenzymes. Other regulatory mechanisms integrate the ATP-generating role of glycolysis with its anabolic roles.

How are dietary sugars other than glucose oxidized?

Other dietary sugars, such as fructose and galactose, are oxidized by conversion to intermediates of glycolysis.

Describe the regulation of PFK-1

Phosphofructokinase-1 (PFK-1) is the rate-limiting enzyme of glycolysis and controls the rate of glucose-6-P entry into glycolysis in most tissues. -PFK-1 is an allosteric enzyme that has a total of six binding sites: two are for substrates (Mg-ATP and fructose-6-P) and four are allosteric regulatory sites (see Fig. 22.12). -The allosteric regulatory sites occupy a physically different domain on the enzyme than the catalytic site. When an allosteric effector binds, it changes the conformation at the active site and may activate or inhibit the enzyme (see also Chapter 9). -The allosteric sites for PFK-1 include an inhibitory site for MgATP, an inhibitory site for citrate and other anions, an allosteric activation site for AMP, and an allosteric activation site for fructose 2,6-bisphosphate (fructose-2,6-bisP) and other bisphosphates. Several different tissue-specific isoforms of PFK-1 are affected in different ways by the concentration of these sub- strates and allosteric effectors, but all contain these four allosteric sites.

Describe the regulation of pyruvate dehydrogenase and its role in regulating glycolysis.

Pyruvate dehydrogenase is also regulated principally by the rate of ATP utilization (see Chapter 20) through rapid phosphorylation to an inactive form. Thus, in a nor- mal respiring cell, with an adequate supply of O2, glycolysis and the TCA cycle are activated together, and glucose can be completely oxidized to CO2. However, when tissues do not have an adequate supply of O2 to meet their ATP demands, the increased NADH/NAD+ ratio inhibits pyruvate dehydrogenase, but AMP activates glycolysis. A proportion of the pyruvate will then be reduced to lactate to allow glycolysis to continue.

Describe the regulation of pyruvate kinase.

Pyruvate kinase exists as tissue-specific isoenzymes. The form present in brain and muscle contains no allosteric sites, and pyruvate kinase does not contribute to the regulation of glycolysis in these tissues. However, the liver isoenzyme can be inhib- ited through phosphorylation by the cAMP-dependent protein kinase, and by a number of allosteric effectors that contribute to the inhibition of glycolysis during fasting conditions. These allosteric effectors include activation by fructose-1,6-bisP, which ties the rate of pyruvate kinase to that of PFK-1, and inhibition by ATP, which signifies high energy levels.

What are the two ways in which cytosolic NADH is deoxidized back to NAD+?

The NADH produced from glycolysis must be continuously reoxidized back to NAD+ to provide an electron acceptor for the glyceraldehyde-3-P dehydrogenase reaction and prevent product inhibition. Without oxidation of this NADH, glycolysis cannot continue. There are two alternate routes for oxidation of cytosolic NADH (Fig. 22.6). -One route is aerobic, involving shuttles that transfer reducing equivalents across the mitochondrial membrane and ultimately to the electron transport chain and oxygen (see Fig. 22.6A). -The other route is anaerobic (without the use of oxygen). In anaerobic glycolysis, NADH is reoxidized in the cytosol by lactate dehydrogenase, which reduces pyruvate to lactate (see Fig. 22.6B).

What is the biphosphoglycerate shunt?

The bisphosphoglycerate shunt is a "side reaction" of the glycolytic pathway in which 1,3-bis-phosphoglycerate is converted to 2,3-bis-phosphoglycerate (2,3- BPG). Red blood cells form 2,3-BPG to serve as an allosteric inhibitor of oxygen binding to heme (see Chapter 44). 2,3-BPG reenters the glycolytic pathway via dephosphorylation to 3-phosphoglycerate. 2,3-BPG also functions as a coenzyme in the conversion of 3-phosphoglycerate to 2-phosphoglycerate by the glycolytic enzyme phosphoglyceromutase. Because 2,3-BPG is not depleted by its role in this catalytic process, most cells need only very small amounts.

What is the central role of glycolysis in fuel metabolism?

The central role of glycolysis in fuel metabolism is related to its ability to generate ATP with, and without, oxygen

What is the Lactate-Cori Cycle?

The cycling of lactate and glucose between peripheral tissues and liver is called Cori cycle.

What happens to the NADH that was generated during glycolysis?

The cytosolic NADH generated via glycolysis transfers its equivalents to mitochondrial NAD+ via shuttle systems across the inner mitochondrial membrane.

Where does glycolysis take place?

The enzymes of glycolysis are in the cytosol.

What biosynthetic pathway does 1,3-BPG contribute to?

The formation of 2,3-BPG.

What biosynthetic pathway does G6P contribute to?

The formation of 5 Carbon sugars

What biosynthetic pathway does Pyruvate contribute to?

The formation of Alanine via Alanine Aminotransferase

What biosynthetic pathway does 3-Phosphoglycerate contribute to?

The formation of Serine

What biosynthetic pathway does Acetyl CoA contribute to?

The formation of fatty acids.

What biosynthetic pathways do the TCA cycle contribute to?

The formation of glutamate and other amino acids.

What biosynthetic pathway does the conversion of G6P to 1,3-BPG contribute to?

The formation of glycerol phosphates

Describe the allosteric inhibition of PFK-1 at the citrate site.

The function of the citrate-anion allosteric site is to integrate glycolysis with other pathways. For example, the inhibition of PFK-1 by citrate may play a role in decreasing glycolytic flux in the heart during the oxidation of fatty acids.

Explain the mechanism of the G3P shuttle.

The glycerol 3-phosphate shuttle is the major shuttle in most tissues. In this shuttle, cytosolic NAD+ is regenerated by cytoplasmic glycerol 3-phosphate dehydrogenase, which transfers electrons from NADH to DHAP to form glycerol 3-phosphate (Fig. 22.7). Glycerol 3-phosphate then diffuses through the outer mitochondrial membrane to the inner mitochondrial membrane, where the electrons are donated to a membrane-bound flavin adenive dinucleofide (FAD)-containing glycerophosphate dehydrogenase. This enzyme, like succinate dehydrogenase, ultimately donates electrons to CoQ, resulting in an energy yield of approximately 1.5 ATP from oxidative phosphorylation. Dihydroxyacetone phosphate returns to the cytosol to continue the shuttle.

Explain tissue-specific modifications of tissues that rely heavily on anaerobic respiration.

Tissues (or cells) that are heavily depend- ent on anaerobic glycolysis usually have a low ATP demand, high levels of gly- colytic enzymes, and few capillaries, such that oxygen must diffuse over a greater distance to reach target cells. The lack of mitochondria, or the increased rate of gly- colysis, is often related to some aspect of cell function. For example, the mature red blood cell has no mitochondria because oxidative metabolism might interfere with its function in transporting oxygen bound to hemoglobin. Some of the lactic acid generated by anaerobic glycolysis in skin is secreted in sweat, where it acts as an antibacterial agent. Many large tumors use anaerobic glycolysis for ATP produc- tion, and lack capillaries in their core.

What modifications must take place in glycolysis for anaerobic respiration to produce as much ATP as aerobic respiration?

To produce the same amount of ATP per unit time from anaerobic glycolysis as from the complete aerobic oxidation of glucose to CO2, anaerobic glycolysis must occur approximately 15 times faster, and use approximately 15 times more glucose. Cells achieve this high rate of glycolysis by expressing high levels of glycolytic enzymes. In certain skeletal muscles and in most cells during hypoxic crises, high rates of glycolysis are associated with rapid degradation of internal glycogen stores to supply the required glucose-6-P.

What happens to 3-PG?

To transfer the remaining low-energy phosphoester on 3-phosphoglycerate to ADP, it must be converted into a high-energy bond. This conversion is accomplished by moving the phosphate to the second carbon (forming 2-phosphoglycerate) and then removing water to form phosphoenolpyruvate (PEP). The enolphosphate bond is a high-energy bond (its hydrolysis releases approximately 14 kcal/mole of energy), so the transfer of phosphate to ADP by pyruvate kinase is energetically favorable (see Fig. 22.5). This final reaction converts PEP to pyruvate.

How is the cycle of glycolysis perpetuated in anaerobic conditions?

Under anaerobic conditions, pyruvate is reduced to lactate by NADH, thereby regenerating the NAD+ required for glycolysis to continue.

Describe the fate of the NADH produced by glycolysis in the cytosol.

● Inner mitochondrial membrane is impermeable to NADH. No transport protein exists that can translocate NADH across this membrane directly. ANSWER: Glycerol-3-P Shuttle is the major shuttle system in most tissue, and takes NADH into the mitochondria.

Describe the structure of lactate dehydrogenase.

● LDH is a tetramer composed of M subunits and H subunits, including M4, M3H1, M2H2, M1H3, and M4 ● M4 facilitates conversion of pyruvate to lactate in SKM, whereas H4 facilitates conversion of lactate to pyruvate in the heart.

When do tissues use anaerobic respiration?

● Limited supply of O2 (kidney medulla, hypoxia) ● Few or no mitochondria (RBC) ● Greatly increased demands for ATP (skeletal muscle during high-intensity exercise)

Describe the structure of phosphofructokinase (PFK-1).

● Six binding sites: Two substrate binding sites: ATP and fructose-6-P. Four allosteric regulatory sites: ATP, citrate, AMP and fructose 2,6-bis-P. ● Consists of three subunits: M (muscle), L (liver) and C (common). Both M and L subunits are sensitive to AMP and ATP regulation, but C subunits are much less so.