The Reactions Of Glycolysis Constitute The Main Pathway Of Glucose Utilization

The overall equation for glycolysis from glucose to lac-tate is as follows:

Glucose + 2ADP + 2Pi ^ 2l(+) - Lactate + 2ATP + 2H2O

All of the enzymes of glycolysis (Figure 17-2) are found in the cytosol. Glucose enters glycolysis by phos-phorylation to glucose 6-phosphate, catalyzed by hexokinase, using ATP as the phosphate donor. Under physiologic conditions, the phosphorylation of glucose to glucose 6-phosphate can be regarded as irreversible. Hexokinase is inhibited allosterically by its product, glucose 6-phosphate. In tissues other than the liver and pancreatic B islet cells, the availability of glucose for

Glucose

Glycogen (C6)n

Hexose phosphates

Hexose phosphates

Triose phosphate

Triose phosphate

Triose phosphate

Triose phosphate

1/2O2

Lactate

Figure 17-1. Summary of glycolysis. (3, blocked by anaerobic conditions or by absence of mitochondria containing key respiratory enzymes, eg, as in erythro-cytes.

1/2O2

Lactate

Figure 17-1. Summary of glycolysis. (3, blocked by anaerobic conditions or by absence of mitochondria containing key respiratory enzymes, eg, as in erythro-cytes.

glycolysis (or glycogen synthesis in muscle and lipogen-esis in adipose tissue) is controlled by transport into the cell, which in turn is regulated by insulin. Hexokinase has a high affinity (low Km) for its substrate, glucose, and in the liver and pancreatic B islet cells is saturated under all normal conditions and so acts at a constant rate to provide glucose 6-phosphate to meet the cell's need. Liver and pancreatic B islet cells also contain an isoenzyme of hexokinase, glucokinase, which has a Km very much higher than the normal intracellular concentration of glucose. The function of glucokinase in the liver is to remove glucose from the blood following a meal, providing glucose 6-phosphate in excess of requirements for glycolysis, which will be used for glycogen synthesis and lipogenesis. In the pancreas, the glucose 6-phosphate formed by glucokinase signals increased glucose availability and leads to the secretion of insulin.

Glucose 6-phosphate is an important compound at the junction of several metabolic pathways (glycolysis, gluconeogenesis, the pentose phosphate pathway, gly-cogenesis, and glycogenolysis). In glycolysis, it is converted to fructose 6-phosphate by phosphohexose-isomerase, which involves an aldose-ketose isomerization.

This reaction is followed by another phosphorylation with ATP catalyzed by the enzyme phosphofructoki-nase (phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction may be considered to be functionally irreversible under physiologic conditions; it is both inducible and subject to allosteric regulation and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are inter-converted by the enzyme phosphotriose isomerase.

Glycolysis continues with the oxidation of glycer-aldehyde 3-phosphate to 1,3-bisphosphoglycerate. The enzyme catalyzing this oxidation, glyceraldehyde 3-phosphate dehydrogenase, is NAD-dependent. Structurally, it consists of four identical polypeptides (monomers) forming a tetramer. —SH groups are present on each polypeptide, derived from cysteine residues within the polypeptide chain. One of the —SH groups at the active site of the enzyme (Figure 17-3) combines with the substrate forming a thiohemi-acetal that is oxidized to a thiol ester; the hydrogens removed in this oxidation are transferred to NAD+. The thiol ester then undergoes phosphorolysis; inorganic phosphate (P;) is added, forming 1,3-bisphosphoglycer-ate, and the —SH group is reconstituted.

In the next reaction, catalyzed by phosphoglycerate kinase, phosphate is transferred from 1,3-bisphospho-glycerate onto ADP, forming ATP (substrate-level phosphorylation) and 3-phosphoglycerate. Since two molecules of triose phosphate are formed per molecule of glucose, two molecules of ATP are generated at this stage per molecule of glucose undergoing glycolysis. The toxicity of arsenic is due to competition of arsenate with inorganic phosphate (Pi) in the above reactions to give 1-arseno-3-phosphoglycerate, which hydrolyzes spontaneously to give 3-phosphoglycerate plus heat, without generating ATP. 3-Phosphoglycerate is isomer-ized to 2-phosphoglycerate by phosphoglycerate mu-tase. It is likely that 2,3-bisphosphoglycerate (diphos-phoglycerate; DPG) is an intermediate in this reaction.

The subsequent step is catalyzed by enolase and involves a dehydration, forming phosphoenolpyruvate. Enolase is inhibited by fluoride. To prevent glycolysis in the estimation of glucose, blood is collected in tubes containing fluoride. The enzyme is also dependent on the presence of either Mg2+ or Mn2+. The phosphate of phosphoenolpyruvate is transferred to ADP by pyruvate kinase to generate, at this stage, two molecules of ATP per molecule of glucose oxidized. The product of the enzyme-catalyzed reaction, enolpyruvate, undergoes spontaneous (nonenzymic) isomerization to pyruvate and so is not available to

Glycogen

Glucose 1-phosphate

Glucose 1-phosphate

H OH a-D-Glucose

ADP H OH

a-D-Glucose 6-phosphate

H OH a-D-Glucose

ADP H OH

OH H D-Fructose 6-phosphate

D-Fructose 1,6-bisphosphate lodoacetate ■

COO-

PHOSPHOGLYCERATE KINASE

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Mg2+

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

NAD+

OH H D-Fructose 6-phosphate

D-Fructose 1,6-bisphosphate lodoacetate ■

ch2oh

Dihydroxyacetone phosphate

ch2oh

Dihydroxyacetone phosphate

3-Phosphoglycerate ch2—o—(p

1,3-Bisphosphoglycerate

NAD+

PHOSPHOGLYCERATE MUTASE

COO-

Fluoride O

Anaerobiosis

COO-

I Phosphoenolpyruvate

COO-

(Enol) Pyruvate

PYRUVATE KINASE

Oxidation in citric acid cycle

COO-

Mitochondrial respiratory chain ch2-o—®

Glyceraldehyde 3-phosphate

PHOSPHOTRlOSE lSOMERASE

1/202

Mitochondrial respiratory chain

3ATP

Spontaneous

(Keto) Pyruvate

LACTATE DEHYDROGENASE

COO-I

Figure 17-2. The pathway of glycolysis. (®, —PO32-; Pi, HOPO32-; ©, inhibition.) At asterisk: Carbon atoms 1-3 of fructose bisphosphate form dihydroxyacetone phosphate, whereas carbons 4-6 form glyceraldehyde 3-phosphate. The term "bis-," as in bisphosphate, indicates that the phosphate groups are separated, whereas diphosphate, as in adenosine diphosphate, indicates that they are joined.

Glyceraldehyde 3-phosphate

1,3-Bisphosphoglycerate

Enzyme-substrate complex

Substrate oxidation by bound NAD+

S Enz

Energy-rich intermediate

Figure 17-3. Mechanism of oxidation of glyceraldehyde 3-phosphate. (Enz, glycer-aldehyde-3-phosphate dehydrogenase.) The enzyme is inhibited by the —SH poison iodoacetate, which is thus able to inhibit glycolysis. The NADH produced on the enzyme is not as firmly bound to the enzyme as is NAD+. Consequently, NADH is easily displaced by another molecule of NAD+.

Energy-rich intermediate

Figure 17-3. Mechanism of oxidation of glyceraldehyde 3-phosphate. (Enz, glycer-aldehyde-3-phosphate dehydrogenase.) The enzyme is inhibited by the —SH poison iodoacetate, which is thus able to inhibit glycolysis. The NADH produced on the enzyme is not as firmly bound to the enzyme as is NAD+. Consequently, NADH is easily displaced by another molecule of NAD+.

undergo the reverse reaction. The pyruvate kinase reaction is thus also irreversible under physiologic conditions.

The redox state of the tissue now determines which of two pathways is followed. Under anaerobic conditions, the reoxidation of NADH through the respiratory chain to oxygen is prevented. Pyruvate is reduced by the NADH to lactate, the reaction being catalyzed by lactate dehydrogenase. Several tissue-specific isoenzymes of this enzyme have been described and have clinical significance (Chapter 7). The reoxidation of NADH via lactate formation allows glycolysis to proceed in the absence of oxygen by regenerating sufficient NAD+ for another cycle of the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. Under aerobic conditions, pyruvate is taken up into mitochondria and after conversion to acetyl-CoA is oxidized to CO2 by the citric acid cycle. The reducing equivalents from the NADH + H+ formed in glycolysis are taken up into mitochondria for oxidation via one of the two shuttles described in Chapter 12.

Tissues That Function Under Hypoxic Circumstances Tend to Produce Lactate (Figure 17-2)

This is true of skeletal muscle, particularly the white fibers, where the rate of work output—and therefore the need for ATP formation—may exceed the rate at which oxygen can be taken up and utilized. Glycolysis in erythrocytes, even under aerobic conditions, always terminates in lactate, because the subsequent reactions of pyruvate are mitochondrial, and erythrocytes lack mitochondria. Other tissues that normally derive much of their energy from glycolysis and produce lactate include brain, gastrointestinal tract, renal medulla, retina, and skin. The liver, kidneys, and heart usually take up lactate and oxidize it but will produce it under hypoxic conditions.

Glycolysis Is Regulated at Three Steps Involving Nonequilibrium Reactions

Although most of the reactions of glycolysis are reversible, three are markedly exergonic and must therefore be considered physiologically irreversible. These reactions, catalyzed by hexokinase (and glucokinase), phosphofructokinase, and pyruvate kinase, are the major sites of regulation of glycolysis. Cells that are capable of reversing the glycolytic pathway (gluconeoge-nesis) have different enzymes that catalyze reactions which effectively reverse these irreversible reactions. The importance of these steps in the regulation of gly-colysis and gluconeogenesis is discussed in Chapter 19.

In Erythrocytes, the First Site in Glycolysis for ATP Generation May Be Bypassed

In the erythrocytes of many mammals, the reaction catalyzed by phosphoglycerate kinase may be bypassed by a process that effectively dissipates as heat the free energy associated with the high-energy phosphate of 1,3-bisphosphoglycerate (Figure 17-4). Bisphospho-glycerate mutase catalyzes the conversion of 1,3-bis-phosphoglycerate to 2,3-bisphosphoglycerate, which is converted to 3-ph osphogly cerate by 2, glycerate phosphatase (and possibly also phosphoglyc-erate mutase). This alternative pathway involves no net yield of ATP from glycolysis. However, it does serve to provide 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for oxygen and so making oxygen more readily available to tissues (see Chapter 6).

THE OXIDATION OF PYRUVATE TO ACETYL-CoA IS THE IRREVERSIBLE ROUTE FROM GLYCOLYSIS TO THE CITRIC ACID CYCLE

Pyruvate, formed in the cytosol, is transported into the mitochondrion by a proton symporter (Figure 12-10). Inside the mitochondrion, pyruvate is oxidatively decar-boxylated to acetyl-CoA by a multienzyme complex that is associated with the inner mitochondrial membrane. This pyruvate dehydrogenase complex is analogous to the a-ketoglutarate dehydrogenase complex of the citric acid cycle (Figure 16-3). Pyruvate is decarboxylated by the pyruvate dehydrogenase component of the enzyme complex to a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamin diph osphate, which in turn reacts with oxidized lipoamide, the prosthetic group of dihydrolipoyl transacetylase, to form acetyl lipoamide (Figure 17-5). Thiamin is vitamin B1 (Chapter 45), and

Glyceraldehyde 3-phosphate

NAD+

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

1,3-Bisphosphoglycerate

PHOSPHOGLYCERATE KINASE

2,3-Bisphosphoglycerate

COO-

3-Phosphoglycerate

2,3-Bisphosphoglycerate

Pyruvate

Figure 17-4. 2,3-Bisphosphoglycerate pathway in erythrocytes.

Pyruvate

Figure 17-4. 2,3-Bisphosphoglycerate pathway in erythrocytes.

in thiamin deficiency glucose metabolism is impaired and there is significant (and potentially life-threatening) lactic and pyruvic acidosis. Acetyl lipoamide reacts with coenzyme A to form acetyl-CoA and reduced lipoamide. The cycle of reaction is completed when the reduced lipoamide is reoxidized by a flavoprotein, dihydrolipoyl dehydrogenase, containing FAD. Finally, the reduced flavoprotein is oxidized by NAD+, which in turn transfers reducing equivalents to the respiratory chain.

Pyruvate + NAD + + CoA ^ Acetyl - CoA + NADH + H+ + CO2

The pyruvate dehydrogenase complex consists of a number of polypeptide chains of each of the three component enzymes, all organized in a regular spatial configuration. Movement of the individual enzymes appears to be restricted, and the metabolic intermediates do not dissociate freely but remain bound to the enzymes. Such a complex of enzymes, in which the sub

Lipoic Acid Pathway Include Tdp

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD+, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; TDP, thiamin diphosphate.)

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD+, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; TDP, thiamin diphosphate.)

strates are handed on from one enzyme to the next, increases the reaction rate and eliminates side reactions, increasing overall efficiency.

Pyruvate Dehydrogenase Is Regulated by End-Product Inhibition & Covalent Modification

Pyruvate dehydrogenase is inhibited by its products, acetyl-CoA and NADH (Figure 17-6). It is also regu lated by phosphorylation by a kinase of three serine residues on the pyruvate dehydrogenase component of the multienzyme complex, resulting in decreased activity, and by dephosphorylation by a phosphatase that causes an increase in activity. The kinase is activated by increases in the [ATP]/[ADP], [acetyl-CoA] /[CoA], and [NADH]/[NAD+] ratios. Thus, pyruvate dehydrogenase—and therefore glycolysis—is inhibited not only by a high-energy potential but also when fatty acids are being oxidized. Thus, in starvation, when free fatty acid

Acetyl-CoA

Acetyl-CoA

Pyruvate

[NADH ]

[ ATP ]

[ NAD+]

[ ADP ]

©

/

____ Dichloroacetate

Pyruvate

Insulin (in adipose tissue)

Figure 17-6. Regulation of pyruvate dehydrogenase (PDH). Arrows with wavy shafts indicate allosteric effects. A: Regulation by end-product inhibition. B: Regulation by interconversion of active and inactive forms.

Insulin (in adipose tissue)

Figure 17-6. Regulation of pyruvate dehydrogenase (PDH). Arrows with wavy shafts indicate allosteric effects. A: Regulation by end-product inhibition. B: Regulation by interconversion of active and inactive forms.

concentrations increase, there is a decrease in the proportion of the enzyme in the active form, leading to a sparing of carbohydrate. In adipose tissue, where glucose provides acetyl CoA for lipogenesis, the enzyme is activated in response to insulin.

Oxidation of Glucose Yields Up to 38 Mol of ATP Under Aerobic Conditions But Only 2 Mol When O2 Is Absent

When 1 mol of glucose is combusted in a calorimeter to CO2 and water, approximately 2870 kJ are liberated as heat. When oxidation occurs in the tissues, approximately 38 mol of ATP are generated per molecule of glucose oxidized to CO2 and water. In vivo, AG for the

ATP synthase reaction has been calculated as approximately 51.6 kJ. It follows that the total energy captured in ATP per mole of glucose oxidized is 1961 kJ, or approximately 68% of the energy of combustion. Most of the ATP is formed by oxidative phosphorylation resulting from the reoxidation of reduced coenzymes by the respiratory chain. The remainder is formed by substratelevel phosphorylation (Table 17-1).

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