Glycolysis

Oxidation of glucose or glycogen to pyruvate and lactate is called glycolysis. This was described by Embeden, Meyerhoff and Parnas. Hence it is also called as Embden Meyerhoff pathway. It occurs virtually in all tissues. Erythrocytes and nervous tissues derive its energy mainly form glycolysis. This pathway is unique in the sense that it can utilize O2 if available (‘aerobic’) and it can function in absence of O2 also (‘anaerobic’)

Aerobic Phase

Aerobic phase includes the conversion of glucose to pyruvate Oxidation is carried out by dehydrogenation and reducing equivalent is transferred to NAD. NADH + H+ in presence of O2 is oxidized in electron- transport chain producing ATP.

• Anaerobic Phase

This phase includes the conversion of Glucose to lactate NADH cannot be oxidized, so no ATP is produced in electron transport chain. But the NADH is oxidized to NAD+ by conversion of pyruvate to Lactate, without producing ATP. Anaerobic phase limits the amount of energy per molecule of glucose oxidized. Hence, to provide a given amount of energy, more glucose must undergo glycolysis under anaerobic as compared to aerobic.

A. Enzymes

Enzymes involved in glycolysis are present in cytoplasm.

SIGNIFICANCE OF THE PATHWAY:

This pathway is meant for provision of energy.
It has importance in skeletal muscle as glycolysis provides ATP even in absence of O2, muscles can survive under anaerobic condition.

REACTIONS OF GLYCOLYTIC PATHWAY

Series of reactions of glycolytic pathway, which degrades glucose/ glycogen to pyruvate/lactate, are discussed below. For discussion and proper understanding, the various reactions can be arbitrarily divided in to four stages.

Stage I

This is preparatory stage, before the glucose molecule can be split; the rather asymmetric glucose molecule is converted to almost symmetrical form fructose 1, 6 bisphosphate in the presence of ATP.

1. Uptake of Glucose by Cells and its phosphorylation

Glucose is freely permeable to Liver cells. In Intestinal mucosa and kidney tubules, glucose is taken up by ‘active’ transport. In other tissues, like skeletal muscle, cardiac muscle, diaphragm, adipose tissue etc. Insulin facilitates the uptake of glucose. Glucose is then phosphorylated to form glucose – 6- Phosphate. The reaction is catalyzed by the specific enzyme glucokinase in liver cells and by nonspecific Hexokinase in liver and extrahepatic tissues.

ATP acts as PO4 donor in the presence of Mg .One high energy PO4 bond is utilized and ADP is produced. The reaction is accompanied by considerable loss of free energy as heat, and hence under physiological conditions is regarded as irreversible.
Glucose 6 phosphate formed is an important compound at the junction of several metabolic pathways like glycolysis, glycogenesis, glycogenolysis, glyconeogenesis, Hexosemonophosphate Shunt, uronic acid partway. Thus is a “committed step” in metabolic pathways.

2. Conversion of G- 6- phosphate to Fructose6-phosphate

• Glucose6 phosphate after formation is converted to fructose 6-p by phospho- hexose isomerase, which involves an aldose- ketose isomerization. The enzyme can act only on α - anomer of Glucose 6 phosphate.

3. Conversion of Fructose 6phosphate to Fructose 1, 6 bisphosphate

The above reaction is followed by another phosphorylation. Fructose-6-p is phosphorylated with ATP at 1- position catalyzed by the enzyme phospho- fructokinase-1 to produce the symmetrical molecule fructose –1, 6 bis phosphate.

Note:

reaction one is irreversible
One ATP is utilized for phosphorylation of glucose at position 6
Phosphofruvctokinase I is the key enzyme in glycolysis that regulates the pathway. The enzyme is inducible, as well as allosterically modified
Phosphofructokinase II is an is enzyme which catalyzes the reaction to form fructose-2 6- bis phosphate.

Fructose-6-phosphate + ATP Fructose-2, 6-bisphosphate + ADP

Energetics

Note that in this stage glucose oxidation does not yield any useful energy rather there is expenditure of 2 ATP molecules for two phosphorylations (-2 ATP).

Stage II

Here,Fructose, 1, 6- bisphosphate is split by the enzyme aldolase into two molecules of triosephosphates, an Aldotriose, glyceraldehyde3 phosphate and a Ketotriose, Dihydroxy acetone phosphate.

Note

The reaction is reversible
There is neither expenditure of energy nor formation ATP
Aldolases are tetramers, containing 4 subunits. Two isoenzymes.A,B Aldolase B: occurs in liver and kidney
The fructose- 6-p exists in the cells in “furanose” form but they react with isomerase, phosphofructokinase-1 and aldolase in the open-chain configuration.
Both triose phosphates are interconvertable

Stage III

This is the energy- yielding reaction. Reactions of this type in which an aldehyde group is oxidized to an acid are accompanied by liberation of large amounts of potentially useful energy.

This stage consists of the following two reactions:

1. Oxidation of Glyceraldehyde 3phosphate to 1,3 bis phosphoglycerate Glycolysis proceeds by the oxidation of glyceraldehde-3-phosphate,to form1,3-bis phosphoglycerate. Dihydroxyacetone phosphate also forms 1, 3 - bisphosphoglycerate via glyceraldehydes-3- phosphate shuttle.The enzyme responsible is Glyceraldehyde 3 phosphate dehydrogenase, which is NAD+ dependant.

Energetics

In first reaction of this stage- NADH produced will be oxidized in electron transport chain to produce 3 ATP in presence of O2. Since two molecules of triose phosphate are formed per molecule of glucose oxidized, 2 NADH will produce 6 ATP.
The second reaction will produce one ATP. Two molecules of substrate will produce ATP.

Net gain at this stage per molecule of glucose oxidized= + 8ATP

Stage IV

This is the recovery of the PO4 group form 3- phosphoglycerate. The two molecules of 3- phosphoglycerate the end- product of the previous stage, still retains the PO4 group originally derived form ATP in stage 1. Body wants to recover the two ATP spent in first stage for two phosphorylation reactions. This is achieved by following three reactions:

Conversion of 3- phosphoglycerate to 2- Phosphoglycerate 3-Phosphoglycerate formed by the above reaction is converted to 2-phosphoglycerate, catalyzed by the enzyme phosphoglycerate mutase. It is likely that 2,3 bisphosphoglycerate is an intermediate in the reaction and probably acts catalytically.
Conversion of 2-phosphoglycerate to Phosphoenol pyruvate The reaction is catalyzed by the enzyme enolase, the enzyme requires the presence of either Mg++ or Mn++ for activity. The reaction involves dehydration and redistribution of energy within the molecule raising the PO4 in position 2 to a “high – energy state”.
Conversion of phosphoenol pyruvate to pyruvate Phosphoenol pyruvate is converted to ‘Enol’ pyruvate, the reaction is catalyzed by the enzyme pyruvate kinase. The high energy PO4 of phosphoenol pyruvate is directly transferred to ADP producing ATP.

Note

Reaction is irreversible
ATP is formed at the substrate level without electron transport chain. This is another example of “ substrate level phosphorylation “ in glycolytic pathway
“Enol“ pyruvate is converted to ‘ Keto’ pyruvate spontaneously.
But, cells having limited coenzymes, to continue the glycolytic cycle NADH must be oxidized to NAD+-. This is achieved by re oxidation of NADH by conversion of pyruvate to lactate (without producing ATP).

Significance of lactate formation:

Under anaerobic conditions NADH is re oxidized via lactate formation. This allows glycolysis to proceed in the absence of oxygen. The process generates enough NAD for another cycle of glycolysis.

B. Clinical Importance

Tissues that function under hypoxic conditions will produce lactic acid from glucose oxidation. Produces local acidosis. If lactate production is more it can produce metabolic acidosis
Vigorously contracting skeletal muscle will produce lactic acid.
Whether O2 is present or not, glycolysis in erythrocytes always terminated in to pyruvate and lactate.

Entry of fructose in to glycolysis:

Liver contains specific enzymes fructokinase. It converts fructose to fructose 1 phosphate in the presence of ATP. In liver fructose1-phosphate is split to glyceraldehyde and dihydroxy acetone phosophate by AldolaseB. Glyceraldehyde enters glycolysis, when it is phosphorylated to glyceraldehyde-3-P by triose kinase. Dihydroxy aceton phosphate and glyceraldehyde-3-P may be degraded via glycolysis or may be condensed to form glucose by aldolase. Lack of fructose kinase leads to fructosuria. Absence of aldolaseB leads to hereditary fructose intolerance. If fructose 1, 6 bisphosphatase is absent, causes fructose induced hypoglycemia. The reason being high concentration of Fructose 1 phosphate and fructose 1, 6 bis phosphate inhibit Liver phosphorylase by allosteric modulation. As in case of Galactose, fructose intolerance can also lead to cataract formation.

Galactose:

Milk sugar contains galactose. Galactokinase converts galatose to galactose-1-P.It reacts with UDP-glucose to form UDP-galactose and glucose-1-P.The enzyme is Galactose-1-P uridyltransferase. UDP-galactose can be epimerized to UDP-glucose by 4- epimerase.Glycogenesis also requires UDP-glucose. UDP-galactose can be condensed with glucose to form lactose.

Galactosemia:

Some people cannot metabolize galactose. It is an inherited disorder that the defect may be in the galactokinase, uridlyl transferase or 4-epimerase.Most common is uridyl transferase. Such patients have high concentration of Galactose in blood (Galactosemia).In lense, Galactose is reduced to galactitol by aldose reductase.The product accumulates in lense and leads to accumulation of water by osmotic pull. This leads to turbidity of lense proteins (Cataract). If uridyl transferase was absent galctose 1-phosphate accumulates.Liver is depleted of inorganic phosphate. This ultimately causes failure of liver function and mental retardation. If 4-epimerase is absent, since the patient can form UDP-galactose from glucose the patient remains symptom free.

Glycogen metabolism

Introduction

Glycogen is the major storage form of carbohydrate in animals .It is mainly stored in liver and muscles and is mobilized as glucose whenever body tissues require.

Degradation of Glycogen (glycogenolysis)

A. Shortening of chains

Golycogen phosphorylase cleaves the α-1, 4 glycosidic bonds between the glucose residues at the non reducing ends of the glycogen by simple phosphorolysis.

This enzyme contains a molecules of covalently bound pyridoxal phosphate required as a coenzyme,
Glycogen phosphorylase is a phosphotransferase that sequentially degrades the glycogen chains at their non reducing ends until four glucose units remain an each chain before a branch point. The resulting structure is called a limit dextrin and phosphorylase cannot degrade it any further. The product of this reaction is Glucose 1 phosphate.
The glucose 1 phosphate is then converted to glucose 6 phosphate by phosphoglucomutase.
Conversion of glucose 6 phosphate to glucose occurs in the Liver, Kidney and intestines by the action of Glucose 6 phosphatase. This does not occur in the skeletal muscle as it lacks the Enzyme.

B. Removal of Branches

A debranching enzyme also called Glucantransferase which contains two activities, Glucantransferase and Glucosidase. The transfer activity removes the terminal 3 glucose residues of one branch and attaches them to a free C4 end of the second branch.The glucose in α-(1,6) linkage at the branch is removed by the action of Glucosidase as free glucose.

C. Lysosomal Degradation of Glycogen

A small amount of glycogen is continuously degraded by the lysosomal enzyme α-(1, 4) glycosidase (acid maltase). The purpose of this pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the cytosol, resulting in a very serious glycogen storage disease called type II (Pomp’s disease)

Fig.2.9 Summary of Glycogenolysis

Synthesis of Glygogen (Glycogenesis)

Synthesis of glycogen from Glucose is carried out by the enzyme Glycogen Synthase.The activation of glucose to be used for glycogen synthesis is carried out by the enzyme UDPglucose pyrophosphorylase. The enzyme exchanges the phosphate on C-1 of glucose-1- phosphate for UDP (Uridinediphosphate). The energy of the phospho glycosyl bond of UDPglucose is utilized by glycogen Synthase to catalize the incorporation of glucose in to Glycogen. UDP is subsequently released from the enzyme.The α-1,6 branches in glucose are produced by amylo-(1,4-1,6) transglycosylase,also termed as branching enzyme.This enzyme transfers a terminal fragment of 6 to 7 glucose residues(from a polymer of atleast 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position.

Fig 2.10. Glycogenesis

Glycogen storage diseases

These are a group of genetic diseases that result from a defect in an enzyme required for either glycogen synthesis or degradation.They result in either formation of glycogen that has an abnormal structure or the accumulation of excessive amounts of normal glycogen in specific tissues, A particular enzyme may be defective in a single tissue such as the liver or the defect may be more generalized, affecting muscle, kidney, intestine and myocardium. The severity of the diseases may range from fatal in infancy to mild disorders that are not life threatening some of the more prevalent glycogen storage diseases are the following.

The Pentose Phosphate Pathway

The pentose phosphate pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are: To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates Enzymes that function primarily in the reductive direction utilize the NADP+ /NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD+ /NADH cofactor pair. The reactions of fatty acid and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione. The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source, therefore, any rapidly proliferating cell needs large quantities of NADPH.

Fig 2.11 Oxidative and non –oxidative phases of HMP shunt

Significance of HMP shunt

The net result of the PPP, if not used solely for R5P production, is the oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar. In turn, 3 moles of 5 carbon sugar are converted, via the enzymes of the PPP, back into two moles of 6 carbon sugars and one mole of 3 carbon sugar. The 6 carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. The 3 carbon sugar generated is glyceraldehyde-3-phsphate which can be shunted to glycolysis and oxidized to pyruvate. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more 6 carbon sugars (fructose-6-phosphate or glucose-6-phosphate). Glutathione is the tripeptide γ-glutamylcysteinylglycine. The cysteine thiol plays the role in reducing oxidized thiols in other proteins. Oxidation of 2 cysteine thiols forms a disulfide bond. Although this bond plays a very important role in protein structure and function, inappropriately introduced disulfides can be detrimental. Glutathione can reduce disulfides nonenzymatically. Oxidative stress also generates peroxides that in turn can be reduced by glutathione to generate water and an alcohol. It can also reduce hydrogen per-oxide in to two molecules of water. Regeneration of reduced glutathione is carried out by the enzyme, glutathione reductase. This enzyme requires the co-factor NADPH when operating in the direction of glutathione reduction which is the thermodynamically favored direction of the reaction. It should be clear that any disruption in the level of NADPH may have a profound effect upon a cells ability to deal with oxidative stress. No other cell than the erythrocyte is exposed to greater oxidizing conditions. After all it is the oxygen carrier of the body. The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival. Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed to be associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent. The basis for this resistance is the weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.

Coris Cycle or Lactic Acid Cycle

In an actively contracting muscle, only about 8% of the pyruvate is utilized by the citric acid cycle and the remaining is, therefore, reduced to lactate. The lactic acid thus generated should not be allowed to accumulate in the muscle tissues. The muscle cramps, often associated with strenuous muscular exercise are thought to be due to lactate accumulation. This lactate diffuses into the blood. During exercise, blood lactate level increases considerably. Lactate then reaches liver where it is oxidized to pyruvate. It is then taken up through gluconeogenesis pathway and becomes glucose, which can enter into blood and then taken to muscle. This cycle is called cori's cycle, by which the lactate is efficiently reutilized by the body.

Significance of the cycle:

Muscle cannot form glucose by gluconeogenesis process because glucose 6 phosphatase is absent. Unlike Liver, muscle cannot supply Glucose to other organs inspite of having Glycogen.

Fig 2.12 The Cori cycle.

Gluconeogenesis

Gluconoegenesis is the biosynthesis of new glucose from non carbohydrate substrates. In the absence of dietary intake of carbohydrate liver glycogen can meet these needs for only 10 to 18 hours During prolonged fast hepatic glycogen stores are depleted and glucose is formed from precursors such as lactate, pyruvate, glycerol and keto acids. Approximately 90% of gluconeogenesis occurs in the liver whereas kidneys provide 10 % of newly synthesized glucose molecules, The kidneys thus play a minor role except during prolonged starvation when they become major glucose producing organs.

Reactions Unique to Gluconeogenesis

Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose from lactate or pyruvate. However three of the reactions are irreversible and must be bypassed by four alternate reactions that energetically favor the synthesis of glucose.

A. Carboxylation of Pyruvate

In gluconeogenesis, pyruvate is first carboxylated by pyruvate Carboxylase to oxaloacetate (OAA).where it is converted to Phosphoenolpyruvate (PEP) by the action of PEP carboxykinase, Note: pyruvate carboxylase is found in the mitochondria of liver and kidneys, but not in muscle

Biotin is a coenzyme of pyruvate carboxylase derived from vitamin B6 covalently bound to the apoenyme through an ε-amino group of lysine forming the active enzyme.
Allosteric regulation

Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA may signal one of several metabolic states in which the increased synthesis of oxaloacetate is required. For example, this may occur during starvation where OAA is used for the synthesis of glucose by gluconeogenesis, At low levels of acetyl COA, pyruvate carboxylase is largely inactive and pyruvate is primarily oxidized in the TCA cycle

B. transport of Oxaloacetate to the Cytosol

Oxaloacetate, formed in mitochondria, must enter the cytosol where the other enzymes of gluconeogenesis are located. However, oxaloacetate is unable to cross the inner mitochondrial membrane directly. It must first be reduced to malate which can then be transported from the mitochondria to the cytosol. In the cytosol, Malate is reoxidized to oxaloactate (see figure 2.13)

C. Decarboxylation of Cytosolic Oxaloacetate.

Oxaloacetate is decarboxylated and phosphorylated in the cytosol by PEP-carboxykinase. The reaction is driven by hydrolysis of GTP The combined action of pyruvate carboxylase and PEP carboxykinase provides an energetically favorable pathway from pyruvate to PEP. PEP then enters the reversed reactions of glycolysis until it forms fructose 1, 6- bisphosphate. (see figure 2.13)

D. Dephosphorylation of fructose 1, 6 bisphosphate

Hydrolysis of fructose 1, 6-bisphosphate by fructose 1, 6-bisphosphatase passes the irreversible PFK- 1 reaction and provides energetically favorable pathway for the formation of fructose 6-phosphate.

This reaction is an important regulatory site of gluconeogenesis,

1. Regulation by energy levels within the cell: Fructose1, 6 bisphatase is inhibited by elevated levels of AMP, which signal an energy poor state in the cell Conversely high levels of ATP and low concentrations of AMP stimulate gluconeogensis

2. Regulation by fructose 2,6- bisphoshate Fructose1, 6-bisphosphatase is inhibited by fructose 2, 6-bisphosphate, an allosteric modifier whose concentration is influenced by the level of circulating glucagons. Fructose 1, 6 bisphos phatase occurs in liver and kidney

E. Dephosphorylation of glucose 6.phosphate

Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction provides energetically favorable pathway for the formation of free glucose, Glucose 6-phosphatase like pyruvate carboxylase, occurs in liver and kidney, but not in muscle.Thus muscle cannot provide blood glucose from muscle glycogen.

C. F. Substrates for Gluconeogenesis

Gluconeogenic precursors are molecules that can give rise to a net synthesis of glucose.They include all the intermediates of glycolysis and the citric acid cycle. Glycerol, lactate, and the α-keto acids obtained from the deamination of glucogenic amino acids are the most important gluconeogenic precursors.

A. Gluconeogenic Precursors

1. Glycerol is released during hydrolysis of triacylgycerol in adipose tissue and is delivered to the liver. Glycerol is phosphorylated to glycerophosphate an intermediate of glycolysis.

2. Lactate is released in the blood by cells, lacking mitochondria such as red blood cells, and exercising skeletal muscle.

B. Ketogenic compounds

AcetylCoA and compounds that give rise to acetyl CoA (for example acetocetate and ketogenic amino acids) cannot give rise to a net synthesis of glucose, this is due to the irreversible nature of the pyruvate dehydrogenase reaction, (pyruvate to acetyl CoA.) These compounds give rise to ketone bodies and are therefore termed Ketogenic.

Advantages of Gluconeogenesis

1) Gluconeogenesis meets the requirements of glucose in the body when carbohydrates are not available in sufficient amounts.

2) Regulate Blood glucose level

3) Source of energy for Nervous tissue and Erythrocytes

4) Maintains level of intermediates of TCA cycle

5) Clear the products of metabolism of other tissues(Muscle)

Fig.2.13 Major control mechanisms affecting glycolysis and gluconeogenesis

Homeostasis of Blood Glucose

Homostasis of glucose is due to balance of addition and utilization of glucose. Fasting blood glucose is maintained between 80-120mg %. After a meal it rises by 40-60mg% and returns to normal within 2-3hours.