Metabolism Of Carbohydrates Biochemistry Notes-II

Carbohydrates Metabolism Biochemistry for Nurses

Fate of Glucose, Glycolysis, Two Phases of Glycolysis, Biomedical Importance of glycolysis, Hexokinase, Glucokinase, Functions of Fluoride.

Fate of Glucose

1:Oxidation

Glycolysis

HMP Shunt

Uronic Acid path way

2:Storage as Glycogen

Glucogenesis

3:Conversion to Fat

Lipogenesis

4:Conversion to Amino Acids

5:Conversion to Other Sugars

Ribose

Fructose

Manose

Galactose

Glycolysis

    Definition: Oxidation of glucose or glycogen to pyruvate and lactate is called glycolysis. This was described by Embden, Meyerhof and Parnas. Hence, it is also called as Embden Meyerhof pathway. Process of fermentation in yeast cells was similar to breakdown of glycogen in muscles. 

    It occurs virtually in all tissues. Erythrocytes and nervous tissues derive its energy mainly from 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).

Two Phases of Glycolysis

    • Aerobic phase: Oxidation is carried out by dehydrogenation and reducing equivalent is transferred to NAD+. Reduced NAD in presence of O2 is oxidised in electron-transport chain producing ATP.

    • Anaerobic phase: NADH cannot be oxidised in electron transport chain, so no ATP is produced in electron transport chain. But the NADH is oxidised to NAD+ by conversion of pyruvate to lactate, without producing ATP. 

    Anaerobic phase limits the amount of energy per mol. of glucose oxidised. Hence, to provide a given amount of energy, more glucose must undergo glycolysis under anaerobic as compared to aerobic.

Enzymes: Enzymes involved in glycolysis are extramitochondrial.

Biomedical Importance

• 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 anoxic episodes.

• Heart muscle: As compared to skeletal muscle, heart muscle is adapted for aerobic performance. It has relatively poor glycolytic activity and poor survival under conditions of ischaemia.

• Role in cancer therapy: In fast-growing cancer cells, rate of glycolysis is very high. Produces more pyruvic acid (PA) than TCA cycle can handle. Accumulation of pyruvic acid leads to excessive formation of lactic acid producing local lactic acidosis. Local acid environment may be congenital for certain cancer therapy. 

• Haemolytic anaemias: Inherited enzyme deficiencies like hexokinase deficiency and pyruvate kinase deficiency in glycolytic pathway enzymes, can produce haemolytic anaemia.

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 into four stages.

Stage I This is a preparatory stage. Before the glucose molecule can be split, the rather asymmetric glucose molecule is converted to almost symmetrical form fructose 1,6- biphosphate by donation of 2 PO4 groups from ATP.

1. Uptake of glucose by cells and its phosphorylation: Glucose is freely permeable to Liver cells. Insulin facilitates the uptake of glucose in skeletal muscles, cardiac muscle, diaphragm and adipose tissue. 

    Glucose is then phosphorylated to form glucose-6-P. The reaction is catalyzed by the specific enzyme glucokinase in liver cells and by non-specific hexokinase in liver and extrahepatic tissues (Refer second box in right hand side this page).

Note 

• Reaction is irreversible – ATP acts as PO4 donor and it reacts as Mg-ATP complex. One high energy PO4 bond is utilised and ADP is produced. 

    The reaction is accompanied by considerable loss of free energy as heat, and hence under physiologic conditions is regarded as irreversible. – Glucose-6-P formed is an important compound at the junction of several metabolic pathways like glycolysis, glycogenesis, glycogenolysis, gluconeogenesis, HMP-Shunt, uronic acid pathway. Thus it is a committed step in metabolic pathways.

2. Conversion of G-6-P to fructose-6-P: G-6-P after formation is converted to fructose-6-P by phosphohexose isomerase, which involves an aldose-ketose isomerisastion. The enzyme can act only on α-anomer of G-6-P.

3. Conversion of fructose-6-P to fructose-1, 6-bi-P: The above reaction is followed by another phosphorylation. Fructose-6-P is phosphorylated with ATP at 1-position catalysed by the enzyme phosphofructokinase-1 to produce the symmetrical molecule fructose-1,6-bi-phosphate.

Note • The reaction is irreversible.

• One ATP is utilised for phosphorylation.

• Phosphofructokinase-1 is the key enzyme in glycolysis which regulates breakdown of glucose. The enzyme is inducible, as well as allosterically modified.

• Phosphofructokinase-2 is an isoenzyme which catalyses the reaction to form fructose-2,6-bi-phosphate. 

Energetic:

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 Actual Splitting of Symmetrical Fructose-1-6-bi-P. Fructose-1,6-bi-P is split by the enzyme aldolase into two

• The fructose-6-P exists in the cells in furanose form but they react with isomerase, phosphofructokinase1 and aldolase in the open-chain configuration

• Both triose phosphates are interconvertible.

 Inhibitors

• Bromohydroxyacetone-P: It resembles structurally to dihydroxyacetone-P. Hence it binds covalently with the γ-COOH group of a glutamate residue of the enzyme phosphotriose isomerase at the active site of the enzyme molecule. 

    Thus the enzyme becomes inactive and cannot catalyse the reaction. It blocks glycolysis at the stage of dihydroxyacetone-P and leads to accumulation of dihydroxyacetone-P and fructose-1,6-bi-phosphate.

Hexokinase	Glucokinase
Non-specific, can phosphorylate any of the hexoses	Specific, can phosphorylate glucose only
More stable	Physiologically more labile
Found almost in all tissues	Found only in liver
Found in foetal as well as in adult liver	Found in adult liver, not in foetal liver
Allosteric inhibition by glucose-6-P	Not inhibited by Glucose-6-P
K is low = 0.1 mM, hence high affinity for glucose	Km is high = 10 mM, low affinity for glucose
Not very much influenced by diabetic state/or fasting	Depressed in fasting and in diabetes  Glucokinase is de in patients of DM, changes according to nutritional status
No change with glucose feeding	Increased by feeding of glucose after fasting
Inhibited by glucocorticoids and GH; insulin does not have effect on hexokinase proteins (isoenzymes)	Inhibited by glucocorticoids and GH; glucose and insulin stimulates.
Hexokinase activity of liver found in three enzyme	Synthesis is induced by insulin, an inducible enzyme Not known
Main function to make available glucose to tissues for oxidation at lower blood glucose level	Main function to clear glucose from blood after meals and at blood levels greater than 100 mg/dl

Stage III

    It 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-3-P to 1,3-bi-phosphoglycerate: Glycolysis proceeds by the oxidation of glyceraldehyde-3-P to form 1,3-bi-phosphoglycerate. Dihydroxyacetone-P also form 1,3-bi-phosphoglycerate via glyceraldehyde-3-P. Enzyme responsible is Glyceraldehyde-3-P dehydrogenase which is NAD+ dependent.

Characteristics of the Enzyme

 • The enzyme is a tetramer, consisting of four identical polypeptides. 

• Four –SH groups are present on each polypeptide derived from cysteine residue in the chain. 

• One of the –SH group forms the “active site” of the enzyme molecule.

2. Conversion of 1,3-Biphosphoglycerate to 3-Phosphoglycerate

    The reaction is catalysed by the enzyme phosphoglycerate kinase. The high energy PO4 bond at position1 can donate the PO4 to ADP and forms ATP molecule. 

    Note: This is a unique example where ATP can be produced at substrate level without participating in electron transport chain. This type of reaction where ATP is formed at substrate level is called as Substrate level phosphorylation.

Inhibitors

• Arsenite If present, it competes with inorganic Pi in the reaction of conversion of glyceraldehyde-3-P to 1,3- biphosphoglycerate and produces 1-arseno-3- phosphoglycerate, which hydrolyses spontaneously to yield 3-phosphoglycerate and heat. 

    Thus in the next step no ATP is produced. This is an important example of the ability of arsenate to uncouple oxidation and phosphorylation.

• Iodoacetate and Iodoacetamide They bind covalently with –SH group and alkylate the –SH group of the enzyme glyceraldehyde-3-P dehydrogenase. They bind irreversibly with the enzyme and inhibits glycolysis. This leads to accumulation of glyceraldehyde-3-P.

Energetics

1. In first reaction of this stage —NADH produced in presence of O2 will be oxidised in electron transport chain to produce 3 ATP. Since two molecules of trioseP are formed per molecule of glucose oxidised 2 NADH will produce 6 ATP.

2. The second reaction will produce one ATP. Two molecules of substrate will produce 2 ATP. + 2 ATP Net gain at this stage per molecule of glucose oxidised = + 8 ATP.

Stage IV

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

1. Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate 3-phosphoglycerate formed by the above reaction is converted to 2-phosphoglycerate, catalysed by the enzyme Phosphoglycerate mutase. It is likely that 2, 3-bi-phosphoglycerate is an intermediate in the reaction and probably acts catalytically.

2. Conversion of 2-Phosphoglycerate to Phosphoenol Pyruvate

The reaction is catalysed 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”.

3. Conversion of Phosphoenol Pyruvate to Pyruvate Phosphoenol pyruvate is converted to ‘Enol’ pyruvate, the reaction is catalysed by the enzyme Pyruvate kinase. The high energy PO4 of phosphoenol pyruvate is directly transferred to ADP producing ATP (Refer box).

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.

Inhibitors

Fluoride inhibits the enzyme enolase.

Clinical Importance

    Sodium fluoride is used along with K-oxalate for collection of blood for glucose estimation. If K-oxalate is used alone, then in vitro glycolysis will reduce the glucose value in the sample.

Functions of Fluoride

• Inhibits in vitro glycolysis by inhibiting enzyme enolase

• Also acts as anticoagulant, and

• Act. as an antiseptic. Energetics: In this stage, 2 molecules of ATP are produced, per molecule of glucose oxidised. + 2 ATP