Carbohydrate Metabolism

Glycolysis

COURSE TITLE: BIOCHEMISTRY 2COURSE CODE: BCHT 202

PLACEMENT/YEAR/LEVEL: 2nd Year/Level 4, 2nd Semester

M.F.Ullah, Ph.D

Cellular Respiration

The living cells obtain energy by oxidizing the nutrients such as glucose through a series of stepwise metabolic reactions. This process is called as cellular respiration.

The cellular respiration can be anaerobic (occuring in the absence of oxygen)Or aerobic (occuring in the presence of oxygen).

Anaerobic respiration involves two major processes:1. Glycolysis2. Lactic acid fermentation

Aerobic respiration involves three major processes:

1.Glycolysis2. TCA cycle3. Oxidative phosphorylation

Aerobic respiration yields more energy than anaerobic respiration

In glycolysis (from the Greek glykys, meaning “sweet,” and lysis, meaning “splitting”), a molecule of glucose is oxidatively degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. Glycolytic pathway operates in the cytosol

Glycolysis was the first metabolic pathway to be elucidated. The complete glycolytic pathway was elucidated by 1940, largely through the pioneering contributions of Gustav Embden, Otto Meyerhof, Carl Neuberg, Jacob Parnas, Otto Warburg, Gerty Cori, and Carl Cori. Glycolysis is also known as the Embden-Meyerhof pathway.

What is Glycolysis ?

The overall equation for glycolysis is:Glucose + 2NAD+ + 2ADP + 2Pi 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

1.Glycolysis is an almost universal central pathway of glucose catabolism. The glycolytic breakdown of glucose is the sole source of metabolic energy in somemammalian tissues and cell types (erythrocytes, renal medulla, brain, and sperm, for example) and partially contributes to the energy requirements of most cell types along with other metabolic pathways.

2. In addition to serving as an anaerobic and aerobic source of ATP, glycolysis is linked to anabolic pathways as it provides biosynthetic precursors. For example, in liver and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosynthesis.

Significance / Purpose

The Reactions of Glycolysis

The glycolytic pathway, which cleaves 1 mole of glucose (6C) to 2 moles of the 3-carboncompound pyruvate, consists of a preparative phase and an ATP-generating phase.

Preparative Phase (requires energy)In the initial preparative phase of glycolysis, glucose is phosphorylated twice by ATP and cleaved into two triose phosphates. The preparative phase thus proceeds withthe investment of 2 ATP.

Energy payoff Phase (releases energy)In the ATP-generating phase, glyceraldehyde 3-phosphate (a triose phosphate) isoxidized by NAD+ and phosphorylated using inorganic phosphate. The high energyphosphate bond generated in this step is transferred to ADP to form ATP. Theremaining phosphate is also rearranged to form another high-energy phosphatebond that is transferred to ADP. Since there are 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.

Glucose metabolism begins with transfer of a phosphate from ATP to glucose to form glucose-6-P. This step is notable for two reasons: (1) glucose 6-phosphate cannot diffuse through the membrane, because of its negative charges, and (2) the addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.

The transfer of the phosphoryl group from ATP to the hydroxyl group on carbon 6 of glucose is catalyzed by hexokinase (In liver this reaction is catalyzed by glucokinase). Kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor.

1. CONVERSION OF GLUCOSE TO GLUCOSE-6 PHOSPHATE

The second step in glycolysis is the isomerization of glucose 6-phosphate to fructose 6-phosphate. Recall that the open chain form of glucose has an aldehyde group at carbon 1, whereas the open-chain form of fructose has a keto group at carbon 2. Thus, the isomerization of glucose 6-phosphate to fructose 6-phosphate is a conversion of an aldose into a ketose. The reaction is catalyzed by phosphoglucose isomerase.

2. ISOMERIZATION OF GLUCOSE 6-PHOSPHATE TO FRUCTOSE 6-PHOSPHATE

A second phosphorylation reaction follows the isomerization step. Fructose 6-phosphate is phosphorylated by ATP to fructose 1,6-bisphosphate (F-1,6-BP). This reaction is catalyzed by phosphofructokinase (PFK).

The prefix bis- in bisphosphate means that two separate monophosphate groups are present, whereas the prefix di- in diphosphate (as in adenosine diphosphate) means that two phosphate groups are present and are connected by an anhydride bond.

3.The Formation of Fructose 1,6-bisphosphate from Fructose 6-phosphate

4. Catalytic Cleavage Six-Carbon Sugar (Fructose 1,6-bisphosphate ) into Two Three-Carbon Fragments (GAP & DHAP)

This reaction takes place with the splitting of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) by the enzyme aldolase. The products of the remaining steps in glycolysis consist of three carbon units rather than six-carbon units

5. Triose phosphate isomerase Salvages a Three-Carbon Fragment

Glyceraldehyde 3-phosphate is on the direct pathway of glycolysis, whereas dihydroxyacetone phosphate is not. Unless a means exists to convert dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, a three-carbon fragment useful for generating ATP will be lost. GAP and DHAP are isomers that can be readily interconverted: dihydroxyacetone phosphate is a ketose, whereas glyceraldehyde 3-phosphate is an aldose. The isomerization of these three-carbon phosphorylated sugars is catalyzed by triose phosphate isomerase

6. Conversion of glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate (1,3-BPG)The preceding steps in glycolysis have transformed one molecule of glucose into two molecules of glyceraldehyde 3- phosphate, but no energy has yet been extracted.

On the contrary, thus far two molecules of ATP have been invested. We come now to a series of steps that harvest some of the energy contained in glyceraldehyde 3-phosphate.

The initial reaction in this sequence is the conversion of glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate (1,3-BPG), a reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase

7.The Formation of ATP from 1,3-Bisphosphoglycerate (substrate level phosphorylation)

The final stage in glycolysis is the generation of ATP from the phosphorylated three-carbon metabolites of glucose. Phosphoglycerate kinase catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3- bisphosphoglycerate to ADP. ATP and 3-phosphoglycerate are the products.

The formation of ATP in this manner is referred to as substrate-level phosphorylation because the phosphate donor, 1,3- BPG, is a substrate with high phosphoryl-transfer potential.Keep in mind that, because of the actions of aldolase and triose phosphate isomerase, two molecules of glyceraldehyde 3- phosphate were formed and hence two molecules of ATP were generated. These ATP molecules make up for the two molecules of ATP consumed in the first stage of glycolysis.

The position of the phosphoryl group shifts in the conversion of 3- phosphoglycerate into 2-phosphoglycerate, a reaction catalyzed by phosphoglycerate mutase. In general, a mutase is an enzyme that catalyzes the intramolecular shift of a chemical group, such as a phosphoryl group.

8. Rearrangement of 3-phosphoglycerate to 2-phosphoglycerate

9. Dehydration of 2-phosphogycerate to Phosphoenolpyruvate

Enolase catalyzes the formation of phosphoenolpyruvate (PEP) by the dehydration of 2-phosphoglycerate. This dehydration markedly elevates the transfer potential of the phosphoryl group.

10. Conversion of Phosphoenolpyruvate to Pyruvate

The last reaction in the glycolytic pathway is the transfer of a phosphoryl group from phosphoenolpyruvate to ADP catalyzed by pyruvate kinase forming the pyruvate .

This is the second substrate level phosphorylation of the pathway yielding ATP. Becausethe molecules of ATP used in forming fructose 1,6-bisphosphate have already been regenerated, the two molecules of ATP generated from phosphoenolpyruvate are "profit."

Substrate level Phosphorylation & the ATP yield in the transformation of glucose into pyruvate :

Substrate level phosphorylation is the mechanism of ATP formation by the transfer of phosphoryl group from the metabolic substrates carrying “high transfer potential phosphoryl group” to ADP. The formation of ATP in the gycolytic pathway is the example of Substrate level phosphorylation.

2 2 ATP

2. The Formation of ATP from Phosphoenol Pyruvate

2 2 ATP

Total = 4 ATP

1. The Formation of ATP from 1,3-Bisphosphoglycerate

Total of 4 ATP is formed when glucose is oxidized to pyruvate in the glycolytic pathway. However the net yield of ATP is 2 after compensating for the 2 ATP utilized in the preparative phase for the conversion of glucose to Fructose 1,6-bisphosphate.

4 ATP formed – 2 ATP utilized = 2 ATP profit

Oxidative Fate of NADH Produced from GlycolysisThe NADH produced from glycolysis must be continuously reoxidized back to NAD+ to provide an electron acceptor for the glyceraldehyde-3-P dehydrogenase reaction. If NADH is not reoxidized to NAD+ glycolysis will stop due to the shortage of NAD+

Without oxidation of this NADH, glycolysis cannot continue. There are two alternate routes for oxidation of cytosolic NADH to NAD+ depending upon aerobic or anaerobic respiration pathways.

One route is aerobic (in the presence of oxygen) , in which oxidation of NADH to NAD+ takes palce across the mitochondrial membrane by the electron transport chain (ETC) and oxygen. The other route is anaerobic (without the use of oxygen). In anaerobic respiration, NADH is reoxidized in the cytosol by lactate dehydrogenase, which reduces pyruvate to lactate (Lactic acid fermentation)

The fate of pyruvate depends on the route used for NADH oxidation.

1. If NADH is reoxidized aerobically by ETC, pyruvate can be used for other pathways, one of which is oxidation to acetyl-CoA and entry into the TCA cycle for complete oxidation yielding more energy. 2. 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 oxidizing the cytoplasmically derived NADH in the electron transport chain and by allowing pyruvate to be oxidized completely to CO2.

Oxidative Fate of Pyruvate Produced from Glycolysis

Anaerobic Glycolysis (Occurs in red blood cells and in exercising muscle cell)When the oxidative capacity of a cell is limited (e.g., the red blood cell, which has no mitochondria and ETC), the pyruvate and NADH produced from glycolysis cannot be oxidized aerobically. The NADH is therefore oxidized to NAD in the cytosol by reduction of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) and called lactic acid fermentation.

ENERGY YIELD OF AEROBIC VERSUS ANAEROBIC RESPIRATION

In both aerobic and anaerobic respiration, each mole of glucose generates 2 moles of ATP, 2 of NADH and 2 of pyruvate by GLYCOLYSIS.

1. The energy yield from anaerobic respiration (glucose to 2 lactate) is only 2 moles of ATP per mole of glucose generated by glycolysis . This is because NADH is recycled to NAD+ by reducing pyruvate to lactate in LDH reaction of lactic acid fermentation. In this reaction neither the NADH nor pyruvate produce energy.

Glucose to 2 pyruvate (Glycolysis)= 2ATP (directly) 2 NADH to 2 NAD+ (Lactic acid fermentation ) = 0 ATP 2 Pyruvate to 2 Lactate (Lactic acid fermentation) = 0 ATP Total yield in anaerobic respiration = 2 ATP

2. However, when oxygen is available (aerobic respiration), and cytosolic NADH can be oxidized by ETC generating ATP and pyruvate can also enter the mitochondria and be completely oxidized to CO2 via Pyruvate dehydrogenase reaction and the TCA cycle giving more ATP.

The oxidation of pyruvate by PDH & TCA cycle route generates roughly 12.5 moles of ATP per mole of pyruvate.

The cytosolic NADH is oxidized by mitochondrial ETC generates approximately 1.5-2.5 moles of ATP per NADH depending upon the transport system that carries NADH electrons to ETC. (glycerol 3–phosphate shuttle (1.5 ATP/NADH) or malate–aspartate shuttle (2.5 ATP/NADH )

Thus, the 2 NADH molecules produced during glycolysis can lead to 3 to 5 molecules of ATP being produced, depending on which transport system is used. Each pyruvate produced can give rise to 12.5 molecules of ATP, altogether 30 to 32 molecules of ATP can be produced from one mole of glucose oxidized to carbon dioxide.

Glucose to 2pyruvate (Glycolysis) = 2ATP (directly)2 NADH to 2 NAD+ (ETC) = 2x1.5 or 2.5= 3 or 5 ATP2 Pyruvate to CO2 (PDH & TCA) = 2x12.5 = 25 ATP Total yield= 30-32 ATP

ACID PRODUCTION IN ANAEROBIC GLYCOLYSIS

Anaerobic glycolysis results in acid production in the form of H+ ions. Glycolysis forms pyruvic acid, which is reduced to lactic acid in the absence of oxygen. 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 eventually diffuse into the blood. If the amount of lactate generated exceeds the buffering capacity of the blood, the pH drops below the normal range (7.4), resulting in lactic acidosis

TISSUES DEPENDENT ON ANAEROBIC GLYCOLYSIS

Many tissues, including red blood cells, the kidney medulla, the tissues of the eye, and skeletal muscles, rely on anaerobic glycolysis for at least a portion of their ATP requirements . The lack of mitochondria, or the increased rate of glycolysis, 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 production, as they lack blood capillaries for oxygen supply in their core.

Fermentation

In yeast and other microorganisms under anaerobic conditions, the NAD+ required for the continuation of glycolysis is regenerated by a process called alcoholic fermentation. In this process pyruvate is converted to acetaldehyde (by pyruvate decarboxylase ) and then to ethanol (by alcohol dehydrogenase) leading to the reoxidation of NADH to NAD+. The energy yeild from alcoholic fermentation is similar to the anaerobic respiration where pyruvate is converted to lactic acid to regenerate NAD+.

The Glycolytic Pathway Is tightly controlled. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs: (1) the production of ATP, generated by the degradation of glucose, and (2) the provision of building blocks for synthetic reactions, such as the formation of fatty acids.

In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites of control. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible; hence, these enzymes would be expected to have regulatory as well as catalytic roles.

Regulation of Glycolysis

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

Bisoynthetic function of Glycolysis

DNA /RNA

Amino acids / Proteins

Lipids

A CASE STUDY

Mrs. Williams recently complaint of nausea, weakness and pain . Her clinical history showed that she suffers from COPD (chronic obstructive pulmonary disease). After a number of diagnostic tests, her family doctor told her that she has lactic acidosis.

Explain the relationship between COPD and lactic acidosis

Animation video: http://www.youtube.com/watch?v=TZE_JEaAOKI