MCAT Biochemistry Review

Chapter 9: Carbohydrate Metabolism I: Glycolysis, Glycogen, Gluconeogenesis, and the Pentose Phosphate Pathway

9.2 Glycolysis

All cells can carry out glycolysis. In a few tissues, most importantly red blood cells, glycolysis represents the only energy-yielding pathway available because red blood cells lack mitochondria, which are required for the citric acid cycle, electron transport chain, oxidative phosphorylation, and fatty acid metabolism (β-oxidation). Glucose is the major monosaccharide that enters the pathway, but others such as galactose and fructose can also be used.

BRIDGE

Red blood cells extrude their mitochondria during development, as discussed in Chapter 7 of MCAT Biology Review. This helps them carry out their function (carrying oxygen) in two ways:

·        Maximizing volume available for hemoglobin, the primary oxygen-carrying protein.

·        Stopping the red blood cell from burning the oxygen it's supposed to be carrying to oxygen-depleted bodily tissues.

Glycolysis is a cytoplasmic pathway that converts glucose into two pyruvates, releasing a modest amount of energy captured in two substrate-level phosphorylations and one oxidation reaction. If a cell has mitochondria and oxygen, the energy-carriers produced in glycolysis (NADH) can feed into the aerobic respiration pathway to generate energy for the cell. If either mitochondria or oxygen is lacking (such as in erythrocytes or exercising skeletal muscle, respectively), glycolysis may occur anaerobically, although some of the available energy is lost.

Glycolysis also provides intermediates for other pathways. In the liver, glycolysis is part of the process by which excess glucose is converted to fatty acids for storage.

IMPORTANT ENZYMES OF GLYCOLYSIS

While glycolysis contains many different steps, as illustrated in Figure 9.2, the MCAT predominantly tests on the enzymes that are highly regulated or that serve an important energetic function. Therefore, we'll focus our attention on five of these enzymes.

Figure 9.2. Glycolysis

REAL WORLD

Because glycolysis is necessary in every cell of the body, there are no known diseases caused by the complete absence of any in glycolysis; in other words, being unable to carry out glycolysis is incompatible with life. Partial enzyme defects are also rare, but include pyruvate kinase deficiency.

Hexokinase and Glucokinase

The first steps in glucose metabolism in any cell are transport across the membrane and phosphorylation by kinase enzymes inside the cell to prevent glucose from leaving via the transporter. Remember from Chapter 2 of MCAT Biochemistry Review that kinases attach a phosphate group from ATP to their substrates. Glucose enters the cell by facilitated diffusion or active transport; in either case, these kinases convert glucose to glucose 6-phosphate. Because the GLUT transporters are specific for glucose (not phosphorylated glucose), the glucose gets “trapped” inside the cell and cannot leak out. Hexokinase is widely distributed in tissues and is inhibited by its product, glucose 6-phosphate. Glucokinase is found only in liver cells and pancreatic β-islet cells; in the liver, glucokinase is induced by insulin. Table 9.1 identifies the differences in their respectiveKm and vmax values. These coincide with the differences in Km values for the glucose transporters in these tissues.

Hexokinase

Glucokinase

Present in most tissues

Present in hepatocytes and pancreatic β-islet cells (along with GLUT 2, acts as the glucose sensor)

Low Km (reaches maximum velocity at low [glucose])

High Km (acts on glucose proportionally to its concentration)

Inhibited by glucose 6-phosphate

Induced by insulin in hepatocytes

Table 9.1. Comparison of Hexokinase and Glucokinase

KEY CONCEPT

Of all the enzymes the MCAT is most likely to test you on, the rate-limiting enzymes for each process are at the top of the list:

·        Glycolysis: phosphofructokinase-1

·        Fermentation: lactate dehydrogenase

·        Glycogenesis: glycogen synthase

·        Glycogenolysis: glycogen phosphorylase

·        Gluconeogenesis: fructose-1,6-bisphosphatase

·        Pentose Phosphate Pathway: glucose-6-phosphate dehydrogenase

Phosphofructokinases (PFK-1 and PFK-2)

Phosphofructokinase-1 (PFK-1) is the rate-limiting enzyme and main control point in glycolysis. In this reaction, fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate using ATP. PFK-1 is inhibited by ATP and citrate, and activated by AMP. This makes sense because the cell should turn off glycolysis when it has sufficient energy (high ATP) and turn on glycolysis when it needs energy (high AMP). Citrate is an intermediate of the citric acid cycle, so high levels of citrate also imply that the cell is producing sufficient energy.

Insulin stimulates and glucagon inhibits PFK-1 in hepatocytes by an indirect mechanism involving PFK-2 and fructose 2,6-bisphosphate, as shown in Figure 9.2. Insulin activates Phosphofructokinase-2 (PFK-2), which converts a tiny amount of fructose 6-phosphate to fructose 2,6-bisphosphate (F 2,6-BP). F 2,6-BP activates PFK-1. On the other hand, glucagon inhibits PFK-2, lowering F 2,6-BP and thereby inhibiting PFK-1. PFK-2 is found mostly in the liver. By activating PFK-1, it allows these cells to override the inhibition caused by ATP so that glycolysis can continue, even when the cell is energetically satisfied. The metabolites of glycolysis can thus be fed into the production of glycogen, fatty acids, and other storage molecules rather than just being burned to produce ATP.

Glyceraldehyde-3-Phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase catalyzes an oxidation and addition of inorganic phosphate (Pi) to its substrate, glyceraldehyde 3-phosphate. This results in the production of a high-energy intermediate 1,3-bisphosphoglycerate and the reduction of NAD+ to NADH. If glycolysis is aerobic, the NADH can be oxidized (indirectly) by the mitochondrial electron transport chain, providing energy for ATP synthesis by oxidative phosphorylation.

BRIDGE

In Chapter 11 of MCAT General Chemistry Review, we learn that oxidation is loss of electrons, and reduction is gain of electrons. While this is true with biomolecules, it may be easier to think of oxidation as increasing bonds to oxygen or other heteroatoms (atoms besides C and H) and reduction as increasing bonds to hydrogen, as discussed in Chapter 4 of MCAT Organic Chemistry Review. Thus, the conversion of NAD+ to NADH is a reduction reaction.

3-Phosphoglycerate Kinase

3-Phosphoglycerate kinase transfers the high-energy phosphate from 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. This type of reaction, in which ADP is directly phosphorylated to ATP using a high-energy intermediate, is referred to as substrate-level phosphorylation. In contrast to oxidative phosphorylation in mitochondria, substrate-level phosphorylations are not dependent on oxygen, and are the only means of ATP generation in an anaerobic tissue.

Pyruvate Kinase

The last enzyme in aerobic glycolysis, it catalyzes a substrate-level phosphorylation of ADP using the high-energy substrate phosphoenolpyruvate (PEP). Pyruvate kinase is activated by fructose 1,6-bisphosphate from the PFK-1 reaction. This is referred to as feed-forward activation, meaning that the product of an earlier reaction of glycolysis (fructose 1,6-bisphosphate) stimulates, or prepares, a later reaction in glycolysis (by activating pyruvate kinase).

FERMENTATION

In the absence of oxygen, fermentation will occur. The key fermentation enzyme in mammalian cells is lactate dehydrogenase, which oxidizes NADH to NAD+, replenishing the oxidized coenzyme for glyceraldehyde-3-phosphate dehydrogenase. Without mitochondria and oxygen, glycolysis would stop when all the available NAD+ had been reduced to NADH. By reducing pyruvate to lactate and oxidizing NADH to NAD+, lactate dehydrogenase prevents this potential problem from developing. There is no net loss of carbon in this process: pyruvate and lactate are both three-carbon molecules. In aerobic tissues, lactate does not normally form in significant amounts. However, when oxygenation is poor (during strenuous exercise in skeletal muscle, a heart attack, or a stroke), most cellular ATP is generated by anaerobic glycolysis, and lactate production increases.

In yeast cells, fermentation is the conversion of pyruvate (three carbons) to ethanol (two carbons) and carbon dioxide (one carbon). While the end products are different, the result of both mammalian and yeast fermentation is the same: replenishing NAD+.

IMPORTANT INTERMEDIATES OF GLYCOLYSIS

Glycolysis serves as a crossroads for a number of metabolic processes; the intermediates of glycolysis are often used to link different pathways during both catabolism and anabolism. Three of these intermediates are worth highlighting:

·        Dihydroxyacetone phosphate (DHAP) is used in hepatic and adipose tissue for triacylglycerol synthesis. DHAP is formed from fructose 1,6-bisphosphate. It can be isomerized to glycerol 3-phosphate, which can then be converted to glycerol, the backbone of triacylglycerols.

·        1,3-Bisphosphoglycerate (1,3-BPG) and phosphoenolpyruvate (PEP) are high-energy intermediates used to generate ATP by substrate-level phosphorylation. This is the only ATP gained in anaerobic respiration.

IRREVERSIBLE ENZYMES

Three enzymes in the pathway catalyze reactions that are irreversible. This keeps the pathway moving in only one direction. However, the liver must be able to generate new glucose from other biomolecules through gluconeogenesis, which is essentially the reverse of glycolysis. Because of the irreversible enzymes of glycolysis, different reactions, and therefore different enzymes, must be used at these three points:

·        Glucokinase or hexokinase

·        PFK-1

·        Pyruvate kinase

MNEMONIC

Irreversible steps of glycolysis:

How Glycolysis Pushes Forward the Process: Kinases.

·        Hexokinase

·        Glucokinase

·        PFK-1

·        Pyruvate Kinase

GLYCOLYSIS IN ERYTHROCYTES

In erythrocytes (red blood cells), anaerobic glycolysis represents the only pathway for ATP production, yielding a net 2 ATP per glucose.

BRIDGE

Adaptation to high altitudes (low pO2) involves:

·        Increased respiration

·        Increased oxygen affinity for hemoglobin (initial)

·        Increased rate of glycolysis

·        Increased [ 2,3-BPG] in RBC (over a 12–24 hour period)

·        Normalized oxygen affinity for hemoglobin restored by the increased level of 2,3-BPG

·        Increased hemoglobin (over days to weeks)

Gas exchange is discussed in Chapter 6 of MCAT Biology Review, and effects on hemoglobin are discussed in Chapter 7 of MCAT Biology Review.

Red blood cells have bisphosphoglycerate mutase, which produces 2,3-bisphosphoglycerate (2,3-BPG) from 1,3-BPG in glycolysis. Remember that mutases are enzymes that move a functional group from one place in a molecule to another; in this case, the phosphate is moved from the 1-position to the 2-position. 2,3-BPG binds allosterically to the β-chains of hemoglobin A (HbA) and decreases its affinity for oxygen. This effect of 2,3-BPG is seen in the oxygen dissociation curve for HbA, shown in Figure 9.3. The rightward shift in the curve is sufficient to allow unloading of oxygen in tissues, but still allows 100 percent saturation in the lungs. An abnormal increase in erythrocyte 2,3-BPG might shift the curve far enough so that HbA is not fully saturated in the lungs.

Figure 9.3. Effect of 2,3-Bisphosphoglycerate on Hemoglobin A

BRIDGE

Remember the other physiological changes that promote a right shift of the oxygen dissociation curve (the Bohr effect), discussed in Chapter 7 of MCAT Biology Review:

·        High 2,3-BPG

·        Low pH

·        High [H+]

·        High pCO2

These all occur during exercise, giving the mnemonic: “Exercise is the right thing to do.”

Although 2,3-BPG binds to HbA, it does not bind well to fetal hemoglobin (HbF), with the result that HbF has a higher affinity for oxygen than maternal HbA, allowing transplacental passage of oxygen from mother to fetus.

MCAT Concept Check 9.2:

Before you move on, assess your understanding of the material with these questions.

1.    What are the function and key regulators of the following enzymes? Which ones are reversible?

o   Hexokinase

·        Function:

·        Regulation:

·        Reversible? 

o   Glucokinase

·        Function:

·        Regulation:

·        Reversible? 

o   Phosphofructokinase-1 (PFK-1)

·        Function:

·        Rgulation:

·        Reversible? 

o   Glyceraldehyde-3-phosphate dehydrogenase

·        Function:

·        Reversible? 

o   3-phosphoglycerate kinase

·        Function:

·        Reversible? 

o   Pyruvate kinase

·        Fnction:

·        Regulation:

·        Reversible? 

2.    Why must pyruvate undergo fermentation for glycolysis to continue?

3.    Why is it necessary that fetal hemoglobin does not bind 2,3-BPG?