1. Step 8: Migration of the Phosphate

2. Step 8: Migration of the Phosphate
Rationale:
Be able to form high-energy phosphate compound
Mutases catalyze the (apparent) migration of functional groups
One of the active site histidines is post-translationally modified to phosphohistidine
Phosphohistidine donates its phosphate to O2 before retrieving another phosphate from O3
2,3-bisphosphoglycerate intermediate
Note that the phosphate from the substrate ends up bound to the enzyme at the end of the reaction

3. Step 8: Migration of the Phosphate
Thermodynamically unfavorable/reversible
Reactant concentration kept high by PGK to push forward

4. Mechanism of Phosphoglycerate Mutase:
Base Catalyzed Phosphoryl Transfer
MECHANISM FIGURE 14-9 (part 1) The phosphoglycerate mutase reaction.

5. Mechanism of Phosphoglycerate Mutase:
Acid Catalyzed Phosphoryl Transfer
MECHANISM FIGURE 14-9 (part 2) The phosphoglycerate mutase reaction.

6. Step 9: Dehydration of 2-PG to PEP

7. Step 9: Dehydration of 2-PG to PEP
Rationale
Generate a high-energy phosphate compound
2-Phosphoglycerate is not a good enough phosphate donor
Two negative charges in 2-PG are fairly close
But loss of phosphate from 2-PG would give a secondary alcohol with no further stabilization
Slightly thermodynamically unfavorable/reversible
Product concentration kept low to pull forward

8. Step 10: 2nd Production of ATP

9. Step 10: 2nd Production of ATP
Rationale
Substrate-level phosphorylation to make ATP
Net production of 2 ATP/glucose
Loss of phosphate from PEP yields an enol that tautomerizes into ketone
Tautomerization
effectively lowers the concentration of the reaction product
drives the reaction toward ATP formation

10. Pyruvate Tautomerization
Drives ATP Production

11. Step 10: 2nd Production of ATP
Pyruvate kinase requires divalent metals (Mg2+ or Mn2+) for activity
Highly thermodynamically favorable/irreversible
Regulated by ATP, divalent metals, and other metabolites

12. 4C. Summary of Glycolysis
Glucose + 2 NAD+ + 2 ADP + 2 Pi  2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
Used:
1 glucose; 2 ATP; 2 NAD+
Made:
2 pyruvate
Various different fates
4 ATP
Used for energy-requiring processes within the cell
2 NADH
Must be reoxidized to NAD+ in order for glycolysis to continue
Glycolysis is heavily regulated
Ensure proper use of nutrients
Ensure production of ATP only when needed

13. 4D. Fates of Pyruvate
FIGURE 14–4 Three possible catabolic fates of the pyruvate formed in glycolysis. Pyruvate also serves as a precursor in many anabolic reactions, not shown here.

14. 5. Glycolysis occurs at elevated rates in tumor cells
Tumor cells have a higher requirement for glucose due to a lower efficiency in energy production from glycolysis.
Complete oxidation of CO2 in healthy cells under aerobic conditions yields ~30 ATP per glucose.
Anaerobic metabolism of glucose in tumor cells yields 2 ATP per glucose.
Glucose transporters and most glycolytic enzymes are overexpressed in tumors versus normal cells.
Inhibitors of glycolytic pathways could be effective anticancer drugs.
BOX 14-1 FIGURE 1 The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. Glucose transporters and most of the glycolytic enzymes are overproduced in tumors. Compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it.

15. Gluconeogenesis

16. 1. The Body’s Glucose Need
In mammals, some tissues depend almost completely on glucose for their metabolic energy. Glucose from the blood is the sole or major fuel source for:
Human brain and nervous system
- Brain requires 120 g/day, more than half that is stored as glycogen in muscles and liver.
Erythrocytes
Testes
Renal medulla
Embryonic tissues
A mechanism for our bodies to produce glucose is crucial.
Even with other sources serving as fuel in low glucose supply, we still need glucose itself to function.

17. 2. Gluconeogenesis: Precursors for Carbohydrates
Notice that mammals cannot convert fatty acids to sugars.
FIGURE 14–16 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle, which we discuss in Chapter 16. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids (see Table 14–4; see also Fig. 18–15). Plants and photosynthetic bacteria are uniquely able to convert CO2 to carbohydrates, using the Calvin cycle (see Section 20.1).

18. 2. Gluconeogenesis: Precursors for Carbohydrates
Animals can produce glucose from sugars or proteins
Sugars: pyruvate, lactate, or oxaloacetate
Protein: from amino acids that can be converted to citric acid cycle intermediates (or glucogenic amino acids)
Animals cannot produce glucose from fatty acids
Product of fatty acid degradation is acetyl-CoA
Cannot have a net conversion of acetyl-CoA to oxaloacetate
Plants, yeast, and many bacteria can do this, thus producing glucose from fatty acids

20. 3. Glycolysis vs. Gluconeogenesis
Glycolysis occurs mainly in the muscle and brain.
Gluconeogenesis occurs mainly in the liver.
FIGURE 14–17 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14–20 illustrates an alternative route for oxaloacetate produced in mitochondria.

21. 3. Glycolysis vs. Gluconeogenesis
Opposing pathways that are both thermodynamically favorable
Operate in opposite direction
end product of one is the starting compound of the other
Reversible reactions are used by both pathways
Irreversible reaction of glycolysis must be bypassed in gluconeogenesis
Highly thermodynamically favorable, and regulated
Different enzymes in the different pathways
Differentially regulated to prevent a futile cycle

22. 4. Three bypass reactions of gluconeogenesis.
Conversion of pyruvate to phosphoenolpyruvate
Conversion of fructose 1,6-bisphosphate to fructose-6-phosphate
Conversion of glucose-6-phosphate to glucose
These bypasses are irreversible steps.

23. 4A. Pyruvate to Phosphoenolpyruvate
Requires two energy-consuming steps
First step, pyruvate carboxylase converts pyruvate to oxaloacetate
Carboxylation using a biotin cofactor
Requires transport out of the mitochondria via malate
Second step, phosphoenolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate
Phosphorylation from GTP and decarboxylation
Occurs in mitochondria or cytosol depending on the organism

24. 4AIa. Synthesis of Oxaloacetate
This reaction occurs in the mitochondria.
Biotin in a cofactor of the enzyme.
FIGURE 14–18a Synthesis of phosphoenolpyruvate from pyruvate. (a) In mitochondria, pyruvate is converted to oxaloacetate in a biotin-requiring reaction catalyzed by pyruvate carboxylase.

25. 4AIb. Biotin is a CO2 Carrier
FIGURE 14–19 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to the enzyme through an amide linkage to the ε-amino group of a Lys residue, forming a biotinylenzyme. The reaction occurs in two phases, which occur at two different sites in the enzyme. At catalytic site 1, bicarbonate ion is converted to CO2 at the expense of ATP. Then CO2 reacts with biotin, forming carboxybiotinyl-enzyme. The long arm composed of biotin and the Lys side chain to which it is attached then carry the CO2 of carboxybiotinylenzyme to catalytic site 2 on the enzyme surface, where CO2 is released and reacts with the pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme. The general role of flexible arms in carrying reaction intermediates between enzyme active sites is described in Figure 16–18 and the mechanistic details of the pyruvate carboxylase reaction are shown in Figure 16–17. Similar mechanisms occur in other biotin-dependent carboxylation reactions, such as those catalyzed by propionyl-CoA carboxylase (see Fig. 17–12) and acetyl-CoA carboxylase (see Fig. 21–1).

26. 4AIc. Oxaloacetate conversion to malate
Mitochondrion
Oxaloacetate has to be converted to malate for it to be transported out of the mitochondrion.
FIGURE 14–19 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to the enzyme through an amide linkage to the ε-amino group of a Lys residue, forming a biotinylenzyme. The reaction occurs in two phases, which occur at two different sites in the enzyme. At catalytic site 1, bicarbonate ion is converted to CO2 at the expense of ATP. Then CO2 reacts with biotin, forming carboxybiotinyl-enzyme. The long arm composed of biotin and the Lys side chain to which it is attached then carry the CO2 of carboxybiotinylenzyme to catalytic site 2 on the enzyme surface, where CO2 is released and reacts with the pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme. The general role of flexible arms in carrying reaction intermediates between enzyme active sites is described in Figure 16–18 and the mechanistic details of the pyruvate carboxylase reaction are shown in Figure 16–17. Similar mechanisms occur in other biotin-dependent carboxylation reactions, such as those catalyzed by propionyl-CoA carboxylase (see Fig. 17–12) and acetyl-CoA carboxylase (see Fig. 21–1).
Cytosol

27. 4AIIa. Oxaloacetate to Phosphoenolpyruvate
FIGURE 14–18b Synthesis of phosphoenolpyruvate from pyruvate. (b) In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase. The CO2 incorporated in the pyruvate carboxylase reaction is lost here as CO2. The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the γ phosphate of GTP.

28. Summary of first bypass reaction.
Pyruvate + ATP + GTP + HCO3- →
PEP + ADP + GDP + Pi + CO2
ΔG’°= 0.9 kJ/mol
The reaction is irreversible due to the ready consumption of PEP, decreasing the amount of product.

29. 4AIII. From Pyruvate to Phosphoenolpyruvate
Isozymes: Two distinct enzymes that catalyze the same reaction. They can have different cellular locations or metabolic roles.
FIGURE 14–20 Alternative paths from pyruvate to phosphoenolpyruvate. The relative importance of the two pathways depends on the availability of lactate or pyruvate and the cytosolic requirements for NADH for gluconeogenesis. The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text). The requirements of ATP for Pyruvate carboxylase and GTP for PEP carboxykinase (see Fig. 14–17) are omitted for simplicity.

30. 4B. Second bypass reaction
Fructose-1-6-bisphosphatase
+ Mg2+
+ H2O
+ Pi
ΔG’°= kJ/mol

31. 4C. Thirds bypass reaction
Glucose-6-phosphatase
+ Mg2+
Glucose-6-phosphate
Glucose
+ H2O
+ Pi
ΔG’°= kJ/mol

32. 5. Gluconeogenesis is expensive
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O 
Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+
Costs 4 ATP, 2 GTP, and 2 NADH but physiologically necessary
Brain, nervous system, and red blood cells generate ATP ONLY from glucose

33. Pentose Phosphate Pathway

34. 1. Pentose Phosphate Pathway
FIGURE 14–21 General scheme of the pentose phosphate pathway. NADPH formed in the oxidative phase is used to reduce glutathione, GSSG (see Box 14–4) and to support reductive biosynthesis. The other product of the oxidative phase is ribose 5-phosphate, which serves as a precursor for nucleotides, coenzymes, and nucleic acids. In cells that are not using ribose 5-phosphate for biosynthesis, the nonoxidative phase recycles six molecules of the pentose into five molecules of the hexose glucose 6-phosphate, allowing continued production of NADPH and converting glucose 6-phosphate (in six cycles) to CO2.

35. 1. Pentose Phosphate Pathway
The main products are NADPH and ribose 5-phosphate
NADPH is an electron donor
Reductive biosynthesis of fatty acids and steroids
Repair of oxidative damage
For certain tissue, a reducing atmosphere (high ratio of NADPH to NADP+ and a high ratio of reduced to oxidized glutathione) helps combat damage by reactive oxygen species.
Ribose-5-phosphate is a biosynthetic precursor of nucleotides
Used in DNA and RNA synthesis
Or synthesis of some coenzymes

36. 2. NADPH regulates partitioning into glycolysis vs
2. NADPH regulates partitioning into glycolysis vs. pentose phosphate pathway
FIGURE 14–28 Role of NADPH in regulating the partitioning of glucose 6-phosphate between glycolysis and the pentose phosphate pathway. When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction (see Fig. 14–21), [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway. As a result, more glucose 6-phosphate is available for glycolysis.