Glycolysis and Gluconeogenesis

Alice Skoumalová

Metabolism of glucose - overview

1. Glycolysis

Glucose:

the universal fuel for human cells

Sources: diet (the major sugar in our diet) internal glycogen stores blood (glucose homeostasis)

Glucose oxidation: after a meal: almost all tissues during fasting: brain, erythrocytes

Glycolysis:

oxidation and cleavage of glucose

ATP generation (with and without oxygen)

all cells

in the cytosol (the reducing equivalents are transferred to the electron-transport chain by the shuttle)

ATP is generated:

1. via substrate-level phosphorylation

2. from NADH

3. from oxidation of pyruvate

Regulation of glycolysis:

1. Hexokinase

2. Phosphofructokinase

3. Pyruvate Kinase

Generation of precursors for biosynthesis:

fatty acids amino acids ribosis-5-P

Anaerobic glycolysis

a limited supply of O2

no mitochondria increased demands for ATP

Lactic acidemia in hypoxia

Phosphorylation of glucose: irreversible

Glucose 6-P: cannot be transported back across the

plasma membrane a precursor for many pathways that

uses glucose

Hexokinases

Glucokinase (liver, β-cell of the pancreas)

high Km

Michaelis-Menten kinetics

1. Conversion of glucose 6-P to the triose phosphates

2. Oxidation and substrate-level phosphorylation

1. Conversion of glucose 6-P to the triose phosphates

• irreversible

• regulation

essential for the subsequent

cleavage

Substrate-level phosphorylation

Substrate-level phosphorylation

2. Oxidation and substrate-level phosphorylation

Summary of the glycolytic pathway:

Glucosis + 2 NAD+ + 2 Pi + 2 ADP

2 pyruvate + 2 NADH + 4 H+ + 2 ATP + 2 H2O

∆G0´ = - 22 kcal (it cannot be reversed without the expenditure of energy!)

Clinical correlations:

Hypoxemia (lack of oxygen in tissues)

Acute hemorrhage (hypotension, lost of erythrocytes)

- anaerobic glycolysis

- lactate formation, metabolic acidosis

Chronic obstructive pulmonary disease (an insuficient ventilation)

- anaerobic glycolysis, lactate formation, metabolic acidosis

- accumulation of CO2, respiratory acidosis

Myocardial infarction (lack of oxygen in myocardium)

- anaerobic glycolysis, lactate formation

- lack of ATP

Aerobic glycolysis:

involving shuttles that transfer reducing equivalents across the mitochondrial membrane

Glycerol 3-phosphate shuttle:

Malate-aspartate shuttle:

Anaerobic glycolysis:

Energy yield 2 mol of ATP

dissociation and formation of H+

Daily lactate production 115 (g/d)

Erythrocytes 29

Skin 20

Brain 17

Sceletal muscle 16

Renal medulla 15

Intestinal mucosa 8

Other tissues 10

Major tissues of lactate production:

(in a resting state)

Cori cycle:

Lactate can be further metabolized by: heart, sceletal muscle

Lactate dehydrogenase: a tetramer (subunits M and H)

Lactate dehydrogenase

Pyruvate + NADH + H+ lactate + NAD+LD

5 isoenzymes:

Heart (lactate)

Muscle (pyruvate)

Biosynthetic functions of glycolysis:

Clinical correlations:

Long-intensity exercise (for example a sprint)

- the need for ATP exceeds the capacity of the mitochondria for oxidative phosphorylation, anaerobic glycolysis

→ lactate formation, muscle fatigue and pain

- a training → the amounts of mitochondria and myoglobin increase

Regulation

Fructose 2,6-bis-phosphate: is not an intermediate of glycolysis!

Phosphofructokinase-2: inhibited through phosphorylation - cAMP-dependent protein kinase (inhibition of glycolysis during fasting-glucagon)

• tissue-specific isoenzymes (low Km, a high afinity)

• glucokinase (high Km)

• the rate-limiting, allosteric enzyme• tissue-specific isoenzymes

the liver isoenzyme - inhibition by cAMP-dependent protein kinase (inhibition of glycolysis during fasting)

Lactic acidemia:

increased NADH/NAD+ ratio inhibition of pyruvate dehydrogenase

2. Gluconeogenesis

Gluconeogenesis:

synthesis of glucose from noncarbohydrate precursors → to maintain blood glucose levels during fasting

liver, kidney

fasting, prolonged exercise, a high-protein diet, stress

Specific pathways:

1. Pyruvate → Phosphoenolpyruvate

2. Fructose-1,6-P → Fructose-6-P

3. Glucose-6-P → Glucose

Precursors for gluconeogenesis

1. lactate (anaerobic glycolysis)

2. amino acids (muscle proteins)

3. glycerol (adipose tissue)

Conversion of pyruvate to phosphoenolpyruvate

1. Pyruvate → Oxaloacetate Pyruvate carboxylase

2. Oxaloacetate → PEP Phosphoenolpyruvate-

carboxykinase

Conversion of phosphoenolpyruvate to glucose

3. Fructose-1,6-P → Fructose-6-P Fructose 1,6-bisphosphatase (cytosol)

4. Glucose-6-P → Glucose Glucose 6-phosphatase (ER)

Clinical correlations:

Alcoholism

- excessive ethanol consumption → increase NADH/NAD+ ratio that drive the lactate dehydrogenase reaction toward lactate

- lack of precursors for gluconeogenesis → its inhibition

- insuficient diet - reduced glucose in the blood, consumption of glycogen in the liver

→ hypoglycemia

3. Regulations

Type Mechanism Example

Substrate concentration Saturation kinetics (according Michaelis-Menten)

Glucokinase (activation after meal- high Km)

Allosteric Conformational changes induced by the binding of the allosteric efector

Enzymes of glycolysis and glukoneogenesis (allosteric effectors ATP, AMP, citrate)

Covalent modification Conformational changes induced by phosphorylation by proteinkinases

Phosphorylation og glycogensynthase and glycogenphosphorylase (glucagon)

Protein-protein interaction

Conformational changes as a result of different protein binding

Muscle glycogenphosphorylase (activated by Ca2+-calmodulin)

Proteolytic cleavage Activation by proteolytic cleavage of precursor molecule

Proteins of coagulation cascade (zymogens)

Enzyme synthesis Induction or repression of the enzyme synthesis

Enzymes of gluconeogenesis (induced during fasting)

Enzyme regulation:

Second messenger - cAMP:

GlucagonAdenylate-cyclase

Regulation of gluconeogenesis: concomitant inactivation of the glycolytic

enzymes and activation of the enzymes of gluconeogenesis

1. Pyruvate → PEPPhosphoenolpyruvate carboxykinase - induced by glucagon, epinephrine, and cortisol

2. Fructose 1,6-P → Fructose 6-PFructose 1,6-bisphosphatase - inhibited by fructose 2,6-P

3. Glucose 6-P → GlucoseGlucose 6-phosphatase - induced during fasting

Summary

Glycolysis• Generation of ATP (with or without oxygen)• The role of glycolysis in different tissues• Lactate production• Regulation (3 key enzymes)

Gluconeogenesis• Activation during fasting, prolonged exercise, after a high-

protein diet• Precursors: lactate, glycerol, amino acids• 3 key reactions: Pyruvate → PEP

Fructose-1,6-P→ Fructose-6-PGlucose-6-P → Glucose

• Regulation

Pictures used in the presentation:

Marks´ Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M. Lieberman, A.D. Marks)