KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Glycolysis and Pyruvate	12
Dehydrogenase

Learning Objectives

Answer questions about carbohydrate digestion

Demonstrate understanding of glucose transport

Understand concepts of aerobic and anaerobic glycolysis

Explain information related to galactose metabolism

Explain information related to fructose metabolism

Answer questions about pyruvate dehydrogenase

OVERVIEW

All cells can carry out glycolysis. In a few tissues—most importantly red blood cells—glycolysis represents the only energy-yielding pathway available. Glucose is the major monosaccharide that enters the pathway, but others such as galactose and fructose can also be used.

The first steps in glucose metabolism in any cell are transport across the membrane and phosphorylation by kinase enzymes inside the cell to prevent it from leaving via the transporter.

CARBOHYDRATE DIGESTION

Only a very small amount of the total carbohydrates ingested are monosaccharides. Most of the carbohydrates in foods are in complex forms, such as starch (amylose and amylopectin) and the disaccharides sucrose and lactose.

•In the mouth, secreted salivary amylase randomly hydrolyzes the starch polymers to dextrins (<8–10 glucoses).

•Upon entry of food into the stomach, the acid pH destroys the salivary amylase.

•In the intestine, the dextrins are hydrolyzed to the disaccharides maltose and isomaltose.

•Disaccharides in the intestinal brush border complete the digestion process:

–Maltase cleaves maltose to 2 glucoses

–Isomaltase cleaves isomaltose to 2 glucoses

–Lactase cleaves lactose to glucose and galactose

–Sucrase cleaves sucrose to glucose and fructose

Uptake of glucose into the mucosal cells is performed by the sodium/glucose transporter, an active transport system.

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Part I ● Biochemistry

Bridge to Physiology

GLUT 4 translocation to the cell membrane in skeletal muscle is stimulated by exercise. This effect, which is independent of insulin, involves a 5′ AMP-activated kinase.

GLUCOSE TRANSPORT

Glucose entry into most cells is concentration driven and independent of sodium. There are 4 major glucose transporters (GLUT), each with a different affinity for glucose coinciding with its respective physiologic role. Normal glucose concentration in peripheral blood is 4–6 mM (70–110 mg/dL).

•GLUT 1 and GLUT 3 mediate basal glucose uptake in most tissues, including brain, nerves, and red blood cells. Their high affinities for glucose ensure glucose entry even during periods of relative hypoglycemia. At normal glucose concentration, GLUT 1 and GLUT 3 are at Vmax.

•GLUT 2, a low-affinity transporter, is in hepatocytes. After a meal, portal blood from the intestine is rich in glucose. GLUT 2 captures the excess glucose primarily for storage. When the glucose concentration

drops below the Km for the transporter, much of the remainder leaves the liver and enters the peripheral circulation. In the β-islet cells of the pancreas. GLUT-2, along with glucokinase, serves as the glucose sensor for insulin release.

•GLUT 4 is in adipose tissue and muscle, and responds to the glucose concentration in peripheral blood. The rate of glucose transport in both these tissues is increased by insulin, which stimulates the movement of additional GLUT 4 transporters to the membrane by a mechanism involving exocytosis.

Decreased insulin decreases the number of plasma membrane GLUT 4 transporters

Cytoplasmic vesicles with membrane-bound GLUT 4 transporters Endocytosis

Increased insulin increases the number of plasma membrane GLUT 4 transporters

Fusion of vesicles

with plasma

membrane Exocytosis

GLUT 4		GLUT 4

Figure I-12-1. Insulin Regulation of Glucose

Transport in Muscle and Adipose Cells

Although basal transport occurs in all cells independently of insulin, the transport rate increases in adipose tissue and muscle when insulin levels rise. Muscle stores excess glucose as glycogen, and adipose tissue requires glucose to form dihydroxyacetone phosphate (DHAP), which is converted to glycerol phosphate used to store incoming fatty acids as triglyceride (TGL, 3 fatty acids attached to glycerol).

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Chapter 12 ● Glycolysis and Pyruvate Dehydrogenase

Table I-12-1. Major Glucose Transporters in Human Cells

	Name			Tissues			Km, Glucose			Functions
	GLUT 1			Most tissues			~1 mM			Basal uptake of glucose
				(brain, red cells)
	GLUT 2		Liver				~15 mM			Uptake and release of
			Pancreatic β-cells							glucose by the liver β-cell
										glucose sensor
	GLUT 3		Most tissues				~1 mM			Basal uptake
	GLUT 4		Skeletal muscle				~5 mM			Insulin-stimulated glucose
			Adipose tissue							uptake; stimulated by
										exercise in skeletal muscle

Normal blood glucose concentration is 4–6 mM (72–110 mg/dL).

			Insulin	Ca2+
		GLUT2		Open Ca2+
	Glucose			channel	Closed KATP
					channel
			Ca2+	Depolarization
		Glucose		of membrane
			ATPases
	Glucokinase				Increased ratio
					of ATP to ADP
	Glycolysis		NAD
		Citric
			ETC	ATP
		Acid			ATP
		Cycle	NADH

Mitochondrion

Note

Glucose induces genetic expression of the insulin gene. Insulin secretion by the pancreatic β-cells is biphasic. Glucose stimulates the first phase (within 15 minutes) with release of preformed insulin. The second phase (several hours) involves insulin synthesis at the gene level.

Figure I-12-2. GLUT2 and Glucokinase Together Function

as the Glucose Sensor in Pancreatic β-Islet Cells

GLYCOLYSIS

Glycolysis is a cytoplasmic pathway that converts glucose into 2 pyruvates, releasing a modest amount of energy captured in 2 substrate-level phosphorylations and 1 oxidation reaction. If a cell has mitochondria and oxygen, glycolysis is aerobic.

If either mitochondria or oxygen is lacking, glycolysis may occur anaerobically (erythrocytes, exercising skeletal muscle), although some of the available energy is lost.

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Part I ● Biochemistry

Insulin

ATP *ADP

Isomerase

+

PFK–2

Fructose 2,

Glucose

Glucose

Glucose-6P

Fructose-6P

Mg2+

ATP ADP

6-bis P

Transport

Hexokinase

*

ATP

Glucokinase (liver)

ADP

PFK–1 +

(phosphofructokinase)

Fructose-1, 6-bis P

Aldolase

Glyceraldehyde-3P

Dihydroxyacetone-P

NAD

(DHAP)

Isomerase

Glycerol-3P

ETC/O2

Pi

Glyceraldehyde 3P

dehydrogenase

Mitochondria

dehydrogenase

NADH

Glycerol-3P

• TGL synthesis

• Electron shuttle

1,3-Bisphosphoglycerate

(RBC)

ADP

Phosphoglycerate

2,3-Bisphosphoglycerate

ATP

kinase

3-Phosphoglycerate

Mutase

2-Phosphoglycerate

Enolase

Pyruvate kinase deficiency

Phosphoenolpyruvate (PEP)

• Hemolytic anemia

ADP

*

• Increased BPG

Pyruvate kinase

• No heinz bodies

NAD

NADH

ATP

–O2

Mitochondria

Cytoplasm

+O2

Pyruvate

Pyruvate dehydrogenase

Pyruvate

Acetyl-CoA

Lactate

Lactate

or

dehydrogenase

TCA

Fatty acid

synthesis

CO2

*Controlled enzymes catalyzing irreversible steps

ATP

Figure I-12-3. Glycolysis

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Chapter 12 ● Glycolysis and Pyruvate Dehydrogenase

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

Important enzymes in glycolysis include:

•Hexokinase/glucokinase

–Glucose entering the cell is trapped by phosphorylation using ATP.

–Hexokinase is widely distributed in tissues, whereas glucokinase is found only in hepatocytes and pancreatic β-islet cells.

–Note below the differences in the Km and Vmax values. These coincide with the differences in Km values for the glucose transporters in these tissues noted earlier.

Table I-12-2. Comparison of Hexokinase and Glucokinase

	Hexokinase			Glucokinase
	Most tissues			Hepatocytes and pancreatic β-islet
				cells (along with GLUT-2, acts as the
				glucose sensor)
	Low Km (0.05 mM in erythrocytes)			High Km (10 mM)
	Inhibited by glucose 6-phosphate			Induced by insulin in hepatocytes

•Phosphofructokinases (PFK-1 and PFK-2)

–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.

–Insulin stimulates and glucagon inhibits PFK-1 in hepatocytes by an indirect mechanism involving PFK-2 and fructose 2,6-bisphosphate.

–Insulin activates PFK-2 (via the tyrosine kinase receptor and activation of protein phosphatases), which converts a tiny amount of fructose 6-phosphate to fructose 2,6-bisphosphate (F2,6-BP).

–F2,6-BP activates PFK-1.

–Glucagon inhibits PFK-2 (via cAMP-dependent protein kinase A), lowering F2,6-BP and thereby inhibiting PFK-1.

–PFK-1 is a multi-subunit enzyme that demonstrates cooperative kinetics.

•Glyceraldehyde 3-phosphate dehydrogenase

–Glyceraldehyde 3-phosphate dehydrogenase catalyzes an oxidation and addition of inorganic phosphate (Pi) to its substrate. 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 reoxidized (indirectly) by the mitochondrial electron transport chain, providing energy for ATP synthesis by oxidative phosphorylation.

Note

Arsenate inhibits the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate by mimicking phosphate in the reaction. The arsenate-containing product is water labile, enabling glycolysis to proceed but resulting in no ATP production.

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Part I ● Biochemistry

•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 a substratelevel phosphorylation.

–Unlike 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, pyruvate kinase 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 (feed-forward activation).

•Lactate dehydrogenase

–Lactate dehydrogenase is used only in anaerobic glycolysis. It reoxidizes 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.

–In aerobic tissues, lactate does not normally form in significant amounts. However, when oxygenation is poor (skeletal muscle during strenuous exercise, myocardial infarction), most cellular ATP is generated by anaerobic glycolysis, and lactate production increases.

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Chapter 12 ● Glycolysis and Pyruvate Dehydrogenase

Glucose Sensing in β-Islet Cells

Similar to hepatocytes of the liver, β-islet cells of the pancreas have GLUT 2 on the plasma membrane to transport glucose into the cells, as well as glucokinase to trap the incoming glucose as glucose 6-phosphate. Because both GLUT 2 and glucokinase have high Km values for glucose, glucose is transported and phosphorylated via first-order kinetics (directly proportional to glucose concentration in the bloodstream).

A 1-day-old infant delivered at 34 weeks’ gestation due to intrauterine growth retardation developed progressive respiratory failure that required intermittent mechanical ventilation. Her blood glucose was 13.4 mM and increased to 24.6 mM. Insulin was administered to normalize her glucose. No C-peptide was detectable. Her parents were second cousins. Both had symptoms of mild diabetes controlled by diet alone. Genetic studies revealed a missense mutation (Ala378Val) in the glucokinase gene. The parents were heterozygous, and the infant homozygous, for the mutation. Recombinant mutant glucokinase showed only 0.02% of the wildtype activity.

Near-complete deficiency of glucokinase activity is associated with permanent neonatal type 1 diabetes. Glucokinase deficiency is the problem in this infant. In contrast to the case above, some mutations in the glucokinase gene alter the Km for glucose. Those mutations which decrease the Km (increasing the affinity for glucose) result in hyperinsulinemia and hypoglycemia. Conversely, mutations which increase the Km (decreasing the affinity for glucose) are associated with some cases of maturity-onset diabetes of the young (MODY).

Important intermediates of glycolysis include the following:

•Dihydroxyacetone phosphate (DHAP) is used in liver and adipose tissue for triglyceride synthesis.

•1,3-bisphosphoglycerate and phosphoenolpyruvate (PEP) are highenergy intermediates used to generate ATP by substrate-level phosphorylation.

Three enzymes in the pathway catalyze reactions that are irreversible. When the liver produces glucose, different reactions and thus different enzymes must be used at these 3 points:

•Glucokinase/hexokinase

•PFK-1

•Pyruvate kinase

Note

C-peptide is a short polypeptide that connects the A-chain to the B-chain in the proinsulin molecule. It is removed after proinsulin is packaged into vesicles in the Golgi apparatus.

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Part I ● Biochemistry

Recall Question

Which of the following transporters increases its uptake of glucose in response to insulin?

A.Glut 1

B.Glut 2

C.Glut 3

D.Glut 4

Answer: D

ATP Production and Electron Shuttles

Anaerobic glycolysis yields 2 ATP/glucose by substrate-level phosphorylation. Aerobic glycolysis yields these 2 ATP/glucose plus 2 NADH/glucose that can be utilized for ATP production in the mitochondria; however, the inner membrane is impermeable to NADH.

Cytoplasmic NADH is reoxidized to NAD and delivers its electrons to one of 2 electron shuttles in the inner membrane. In the malate shuttle, electrons are passed to mitochondrial NADH and then to the electron transport chain. In the glycerol phosphate shuttle, electrons are passed to mitochondrial FADH2.

•Cytoplasmic NADH oxidized using the malate shuttle produces a mitochondrial NADH and yields approximately 3 ATP by oxidative phosphorylation.

•Cytoplasmic NADH oxidized by the glycerol phosphate shuttle

produces a mitochondrial FADH2 and yields approximately 2 ATP by oxidative phosphorylation.

Glycolysis in the Erythrocyte

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

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Chapter 12 ● Glycolysis and Pyruvate Dehydrogenase

α

α

+ 2,3-bisphosphoglycerate

α α

β

β

(2,3-BPG)

β

β

HbA

HbA

2,3-BPG

100

saturation

+

2,3

- BPG

50

HbA

2

O

%

20

40

60

80

100

120

pO2 (mm Hg)

Figure I-12-4. Effect of 2,3-Bisphosphoglycerate on Hemoglobin A

Erythrocytes have bisphosphoglycerate mutase, which produces 2,3-bispho- sphoglycerate (BPG) from 1,3-BPG in glycolysis.

•2,3-BPG binds 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. The rightward shift in the curve is sufficient to allow unloading of oxygen in tissues, but still allows 100% saturation in the lungs.

•An abnormal increase in erythrocyte 2,3-BPG might shift the curve far enough so HbA is not fully saturated in the lungs.

•Although 2,3-BPG binds to HbA, it does not bind well to HbF (α2γ2), with the result that HbF has a higher affinity for oxygen than maternal HbA, allowing transplacental passage of oxygen from mother to fetus.

Pyruvate kinase deficiency is the second most common genetic deficiency that causes a hemolytic anemia (glucose 6-phosphate dehydrogenase, G6PDH, is the most common). Characteristics include:

•Chronic hemolysis

•Increased 2,3-BPG and therefore a lower-than-normal oxygen affinity of HbA

•Absence of Heinz bodies (Heinz bodies are more characteristic of G6PDH deficiency)

The red blood cell has no mitochondria and is totally dependent on anaerobic glycolysis for ATP. In pyruvate kinase deficiency, the decrease in ATP causes the erythrocyte to lose its characteristic biconcave shape and signals its destruction in the spleen. In addition, decreased ion pumping by Na+/K+-ATPase results in loss of ion balance and causes osmotic fragility, leading to swelling and lysis.

Bridge to Physiology

Adaptation to high altitudes (low PO2) involves:

•Increased respiration

•Respiratory alkalosis

•Lower P50

•for hemoglobin (initial)

•Increased rate of glycolysis

•Increased [2,3-BPG] in RBC (12–24 hours)

•Normal P50 for hemoglobin restored by the increased level of 2,3-BPG

•Increased hemoglobin and hematocrit (days–weeks)

Clinical Correlate

Transfused blood has lower than the expected 2,3-BPG levels, making it less efficient at delivering oxygen to peripheral tissues.

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