Glycolysis : Steps, Process, Diagram and enzymes involved

Glycolysis :

Steps, Process, Diagram and enzymes involved

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• Glycolysis is derived from the Greek words (glycose- sweet or sugar; lysis - dissolution). It is a universal pathway in the living cells. The complete pathway of glycolysis was elucidated in 1940. This pathway is often referred to as Embden-Meyerhof pathway (E.M. pathway) in honour of the two biochemists who made a major contribution to the knowledge of glycolysis. 
• Glycolysis is defined as the sequence of reactions converting glucose (or glycogen) to pyruvate or lactate, with the production of ATP. 

Glycolysis : Features 

1. Glycolysis takes place in all cells of the body. The enzymes of this pathway are present in the Cytosomal fraction of the cell. 

2. Glycolysis occurs both in the absence of oxygen (anaerobic) or in the presence of oxygen (aerobic). 

Lactate is the end product under anaerobic condition. 

In the aerobic condition, pyruvate is formed, which is then oxidized to CO2 and H2O. 

3. Glycolysis is a major pathway for ATP synthesis in tissues lacking mitochondria, e.g. erythrocytes, cornea, lens etc. 

4. Glycolysis is very essential for brain which is dependent on glucose for energy. The glucose in brain has to undergo glycolysis before it is oxidized to CO2 and H2O. 

5. Glycolysis (anaerobic) may be summarized by the net reaction:-

           Glucose + 2ADP + 2Pi  --> 2Lacatate + 2ATP

6. Glycolysis is a central metabolic pathway with many of its intermediates providing branch point to other pathways. Thus, the intermediates of glycolysis are useful for the synthesis of amino acids and fat. 

7. Reversal of Glycolysis along with the alternate arrangements at the irreversible steps, will result in the synthesis of glucose (Gluconeogenesis). 

Glycolysis : Sequence Of Reactions

Glycolysis : Sequence Of Reactions

The pathway is divided into three phases:-

1. Energy investment phase or priming stage 
2. Splitting phase 
3. Energy generation phase. 

1. Energy investment phase 

STEP 1.) Glucose is phosphorylated to glucose 6-phosphate by hexokinase or glucokinase (both are isoenzymes). This is an controlling PFK and, ultimated Mg 2+. The enzyme hexokinase is present in almost all the tissues. It catalyses the phosphorylation of various hexoses (fructose, mannose etc.), has low Km for substrates (about 0.1 mM) and is inhibited by glucose 6-phosphate. 

Glucokinase present in liver, catalyses the phosphorylation of only glucose, has high Km for glucose (10 mM) and is not inhibited by glucose 6-phosphate.

Due to high affinity (low Km ), glucose is utilized by hexokinase even at low concentration, whereas glucokinase acts only at higher levels of glucose i.e., after a meal when blood glucose concentration is above 100 mg/dl. 

Glucose 6-phosphate is impermeable to the cell membrane. It is a central molecule with a variety of metabolic fates — glycolysis, glycogenesis, gluconeogenesis and pentose phosphate pathway. 

STEP -2.) Glucose 6-phosphate undergoes isomerization to give fructose 6- phosphate in the presence of the enzyme phosphohexose isomerase and Mg 2+. 

STEP -3.) Fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase (PFK). This is an irreversible and a regulatory step in glycolysis. 

2. Splitting phase 

STEP -4.) The six carbon fructose 1,6-bisphosphate is split (hence the name 

glycolysis) to two three-carbon compounds, glyceraldehyde 3- 

phosphate and dihydroxyacetone phosphate by the enzyme aldolase 

(fructose 1,6-bisphosphate aldolase). 

STEP -5.) The enzyme phosphotriose isomerase catalyses the reversible 

interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone 

phosphate. Thus, two molecules of glyceraldehyde 3-phosphate are 

obtained from one molecule of glucose. 

3. Energy generation phase 

STEP -6.) Glyceraldehyde 3-phosphate dehydrogenase converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. This step is important as it is involved in the formation of NADH + H+ and a high energy compound 1,3-bisphosphoglycerate. Iodoacetate and arsenate inhibit the enzyme glyceraldehyde 3-phosphate dehydrogenase. In aerobic condition,NADH passes through the electron transport chain and 6 ATP (2×3 ATP) are synthesized by oxidative phosphorylation. 

STEP -7.) The enzyme phosphoglycerate kinase acts on 1,3-bisphosphoglycerate resulting in the synthesis of ATP and formation of 3- phosphoglycerate. This step is a good example of substrate level phosphorylation, since ATP is synthesized from the substrate without the involvement of electron transport chain. Phosphoglycerate kinase reaction is reversible, a rare example among the kinase reactions. 

STEP -8.) 3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase. This is an isomerization reaction.

STEP -9.) The high energy compound phosphoenol pyruvate is generated from 2-phosphoglycerate by the enzyme enolase. This enzyme requires Mg2+or Mn2+ and is inhibited by fluoride. For blood glucose estimation in the laboratory, fluoride is added to the blood to prevent glycolysis by the cells, so that blood glucose is correctly estimated. (Fluoride combines with Mg2+ and phosphate to form a complex that binds with active site of enolase and blocks access of substrate. Thus, fluoride is an unusual competitive inhibitor).

STEP -10.) The enzyme pyruvate kinase catalyses the transfer of high energy phosphate from phosphoenol pyruvate to ADP, leading to the formation of ATP. This step also is a substrate level phosphorylation. This reaction is irreversible.

Conversion of pyruvate to lactate—significance

Under anaerobic conditions (lack of O2), pyruvate is reduced by NADH tolactate in presence of the enzyme lactate dehydrogenase (competitive inhibitor - oxamate). The NADH utilized in this step is obtained from the reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase. The formation of lactate allows the regeneration of NAD+ which can be reused by glyceraldehyde 3-phosphate dehydrogenase so that glycolysis proceeds even in the absence of oxygen to supply ATP. The occurrence of uninterrupted glycolysis is very essential in skeletal muscle during strenous exercise where oxygen supply is very limited. Glycolysis in the erythrocytes leads to lactate production, since mitochondria—the centres for aerobic oxidation—are absent. Brain, retina, skin, renal medulla and gastrointestinal tract derive most of their energy from glycolysis.

Production of ATP in glycolysis

Production of ATP in glycolysis

 ATP generation in glycolysis (from glucose) :-

Under Anaerobic conditions, 2 ATP are synthesized while, under Aerobic conditions, 8 or 6 ATP are synthesized—depending on the shuttle pathway that operates.

When the glycolysis occurs from glycogen, one more ATP is generated. This is because no ATP is consumed for the activation of glucose (glycogen directly produces glucose 1-phosphate which forms glucose 6-phosphate). Thus, in anaerobic glycolysis, 3 ATP are produced from glycogen.

Glycolysis and shuttle pathways

In the presence of mitochondria and oxygen, the NADH produced in glycolysis can participate in the shuttle pathways for the synthesis of ATP. If the cytosolic NADH uses malate-aspartate shuttle, 3 ATP are generated from each molecule of NADH. This is in contrast to glycerolphosphate shuttle that produces only 2 ATP. 

Cancer and glycolysis 

Cancer cells display increased uptake of glucose, and glycolysis. As the tumors grow rapidly, the blood vessels are unable to supply adequate oxygen, and thus a condition of hypoxia exists. Due to this, anaerobic glycolysis predominantly occurs to supply energy. 

Irreversible steps in glycolysis

Most of the reactions of glycolysis are reversible. However, the three steps catalysed by the enzymes hexokinase (or glucokinase), phosphofructokinase and pyruvate kinase, are irreversible. These three stages mainly regulate glycolysis. The reversal of glycolysis, with alternate arrangements made at the three irreversible stages, leads to the synthesis of glucose from pyruvate (gluconeogenesis).

Regulation of glycolysis

The three enzymes namely Hexokinase (and glucokinase), Phosphofructokinase and Pyruvate kinase, catalysing the irreversible reactions regulate glycolysis. Hexokinase is inhibited by glucose 6-phosphate. This enzyme prevents the accumulation of glucose 6-phosphate due to product inhibition. Glucokinase, which specifically phosphorylates glucose, is an inducible enzyme. The substrate glucose, probably through the involvement of insulin, induces glucokinase.

Phosphofructokinase (PFK) is the most important regulatory enzyme in glycolysis. This enzyme catalyses the rate limiting committed step. PFK is an allosteric enzyme regulated by allosteric effectors. ATP, citrate and H+ ions (low pH) are the most important allosteric inhibitors, whereas, fructose 2,6-bisphosphate, ADP, AMP and Pi are the allosteric activators.

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