Glycolysis: Introduction, Features, Reactions, Energy yield, Regulation, Lactic acidosis

Glycolysis

• Carbohydrates are the major source of energy for living cells. The major function of carbohydrates in metabolism is as a fuel to be oxidized and provide energy for other metabolic processes.
• The 3 principal monosaccharides resulting from digestive processes are glucose, fructose, and galactose.
• However, Glucose is the central molecule in carbohydrate metabolism since all the major pathways of carbohydrate metabolism are connected with it.

What is Glycolysis?

• Glycolysis is derived from the  Greek words (glycose – sweet or sugar and lysis – dissolution). 
• Glycolysis the sequence of 10 enzyme-catalyzed reactions that converts glucose into pyruvate with the simultaneous production of ATP.
• lt is a  universal pathway in the living cells.
• It is frequently referred to as Embden-Meyerhof-Parnas or EMP pathway, in honor of these pioneer workers i.e Gustave Embden(1874-1933), Otto Meyerhof (1883-1951)  and Jacob Parnas in the field.
• Glycolysis takes place in all cells of the body. The enzymes of this pathway are present in the cytosomal fraction of the cell.

Salient feature of Glycolysis:

• Glycolysis is the central pathway of glucose catabolism.
• It takes place in all cells of the body. The enzymes of this pathway are present in the cytosomal fraction of the cell.
• It is a  major pathway for  ATP synthesis  in  tissues  lacking mitochondria,  e.g. erythrocytes,  cornea, lens  etc.
• Glycolysis  occurs  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.
• The  glucose in  brain has  to  undergo  glycolysis  before  it  is oxidized  to  CO₂ and H₂O so glycolysis  is  very essential  for  brain which  is  dependent  on glucose  for energy.
• 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.
• Reversal  of  glycolysis along  with  the alternate  arrangements at  the  irreversible steps,  will  result in  the  synthesis  of  glucose (gluconeogenesis).

Reactions of Glycolysis:

Fig: 10 steps involved in Glycolysis

During glycolysis, the 6-carbon glucose is broken down into two moles of 3-carbon pyruvate via 10 enzyme-catalyzed sequential reactions as shown in the above figure.

• Glucose  is  phosphorylated  at C₆ to yeild Glucose 6-phosphate  by  Hexokinase  or Glucokinase  (both  are  isoenzymes). This  is  an  irreversible  reaction, dependent on  ATP  and  Mg²⁺.  The enzyme  Hexokinase  is  present  in  almost all  the  tissues.  lt  catalyses  the phosphorylation  of  various  hexose  and  is inhibited  by Glucose  6-phosphate. Glucokinase  present  in  liver,  catalyses the  phosphorylation  of  only  glucose.

• Glucose 6-phosphate is reversibly isomerized to Frucose 6-phosphate by Phosphoglucoisomerase in presence of Mg²⁺.This reaction involves a shift in the carbonyl oxygen from C₁ to C₂, thus converting an aldose into a ketose.

• Fructose 6-phosphate is phosphorylated by ATP to produce Fructose 1, 6-bisphosphate in the presence of another inducible allosteric enzyme Phosphofructokinase (PFK). The enzyme catalyzes the transfer of a phosphate group from ATP to Fructose 6-phosphate at C₁ to yield Fructose 1, 6-bisphosphate. This  is  an irreversible and a  regulatory  step  in  glycolysis.

• Fructose 1, 6-bisphosphate is a molecule with phosphate group on both ends, it splits in the middle into two different triose phosphates, Glyceraldehyde 3-phoshpate(an aldose) and Dihydroxyacetone phosphate(a ketose) so named as Glycolysis. This reaction is catalyzed by the enzyme Fructose bisphosphate aldolase (simply called aldolase) which cleaves the Fructose 1, 6-bisphosphate molecule between C₃ and C₄. Carbon atoms 4, 5 and 6 appear in glyceraldehyde 3-phosphate and 1, 2 and 3 in dihydroxyacetone phosphate.

• 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.

• Glyceraldehyde  3-phosphate  dehydrogenase  converts  Glyceraldehyde 3-phosphate  to 1,3-Bisphosphoglycerate by dehydrogenation. This  step  is  important  as  it is  involved  in the  formation  of NADH⁺ H⁺  and  a high energy  compound  1,3-Bisphosphoglycerate.  lodoacetate  and  Arsenate inhibit  the  enzyme  Glyceraldehyde 3-phosphate  dehydrogenase. ln  aerobic condition, NADH  passes  through the Electron  Transport  Chain  and  6  ATP (2  x  3 ATP)  are  synthesized  by oxidative phosphorylation.

• This is the first ATP-generating reaction in glycolysis. It involves the transfer of high-energy phosphate group from the carboxylic group of 3-Phosphoglyceroyl phosphate to ADP by the enzyme Phosphoglycerate kinase, thus producing ATP and leaving                  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

• The 3-Phosphoglycerate is converted into 2-Phosphoglycerate due to the intramolecular shift of phosphoryl group from C₃ to C₂, by the enzyme Phosphoglycerate mutase. This  is  an  isomerization  reaction.

• The high energy compound  Phosphoenolpyruvate is generated by dehydration of 2-Phosphoglycerate  by the enzyme Enolase.  This  enzyme  requires  Mg²⁺ or Mn²⁺  and  is  inhihited  by  Fluoride.

1. Phosphoenolpyruvate (PEP) is converted into pyruvate in enol form (i.e. enolpyruvate) by the inducible allosteric enzyme Pyruvate kinase(PK). The enzyme catalyzes the transfer of a phosphoryl group from PEP to ADP, thus forming ATP. Pyruvate  kinase requires  K+  and  either  Mg²⁺  or Mn²⁺ . This is the second ATP-generating reaction in glycolysis. This step  also  is  a  substrate  level phosphorylation. This reaction  is irreversible.

The overall reaction of Glycolysis can be summarized as:

Glucose + 2NAD⁺ + 2ADP + 2P_i = 2Pyruvate + 2[NADH⁺+H⁺] + 2ATP + 2H₂O

Thus, three things happen simultaneously in glycolysis :

(a) Glucose is oxidized to pyruvate.

(b) NAD+ is reduced to NADH.

(c) ADP is phosphorylated to form ATP.

There can be no EMP pathway without all 3 events which means that NAD⁺, ADP, and Pi, as well as glucose, must be present.

Muscle or Anaerobic Glycolysis:

• The fate  of  pyruvate  produced  in  glycolysis depends  on  the presence  or  absence  of oxygen in  the  cells.
• Under aerobic conditions, the pyruvate is the product of glycolysis and NADH, formed by the dehydrogenation of glyceraldehyde 3-phosphate, is then reoxidized to NAD⁺ by oxygen.
• Under anaerobic conditions, the NADH generated in glycolysis cannot be reoxidized by oxygen but must be reoxidized to NAD+ by the pyruvate itself, converting pyruvate into lactate. The reaction is catalyzed by Lactate  dehydrogenase.
• Such type of glycolytic sequence actively occur in contracting skeletal muscles   during  strenuous exercise  where oxygen supply is  very  limited. Glycolysis in  the  erythrocytes  leads to  lactate production,  since  mitochondria, the  centres  for aerobic  oxidation are  absent.
• Besides these a large number of microorganisms, the lactic acid bacteria (Lactobacilli, Bacilli, Streptococci and Clostridia) also follow the same path for the reduction of pyruvate to lactate. Such type of fermentation that yields lactate as the sole product is termed homolactic fermentation.

Fig: Anaerobic conversion of Pyruvate into Lactate

Energy Yield in Glycolysis:

The  details  of  ATP generation  in  glycolysis (from  glucose)  are given in the table given below. Under anaerobic  conditions,  2  ATP are  synthesized while,  under  aerobic conditions,  8 ATP are synthesized.

Note:

(-) sign refers to the utilization of ATP in the reaction

(*) NADH so produced(6) or utilized(anaerobic glycolysis) undergo ETC to produce ATP i.e. 1NADH=3ATP

Regulation of Glycolysis:

The three  enzymes  namely  hexokinase  (glucokinase),  phosphofructokinase  and pyruvatekinase,  catalysing  the  irreversible reactions regulate  glycolysis.

Phosphofructokinase:

• Phosphofructokinase is the most important control element in glycolytic pathway.
• It is an allosteric enzyme that catalyzes the rate limiting step.
• The enzyme is regulated by allosteric effecters.
• High levels of ATP allosterically inhibit the enzyme in the liver, thus lowering its affinity for fructose 6-phosphate. Other allosteric inhibitors are citrate  and  H+ ions  (low pH).
• Fructose  2,6-bisphosphate,  ADP, AMP and Pi are  the allosteric  activators.

Hexokinase:

• It is  inhibited  by  glucose 6-phosphate.
• This  enzyme  prevents  the accumulation  of  glucose 6-phosphate  due  to product  inhibition. High concentrations of this molecule signal that the cell no longer requires glucose for energy, and the glucose will be left in the blood.
• Glucokinase,  which specifically  phosphorylates glucose,  is  an inducible  enzyme.  The  substrate  glucose, probably  through  the  involvement  of  insulin, induces  glucokinase.

Pyruvate kinase:

• Pyruvate kinase also regulates glycolysis.
• This enzyme is inhibited by  ATP and activated by fructose 1,6-bisphosphate.

Note:

Phosphofructokinase has a greater regulatory effect than Hexokinase. The reason becomes evident as glucose 6-phosphate is not solely a glycolytic intermediate. Glucose 6-phosphate can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway to form NADPH. The first irreversible reaction unique to the glycolytic pathway, the committed step, is the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Thus, it is highly appropriate for phosphofructokinase to be the primary control site in glycolysis. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway.

Lactic acidosis:

• Lactic acidosis is a form of metabolic acidosis that begins in the kidneys. People with lactic acidosis have kidneys that are unable to remove excess acid from their body.
• Lactic  acid  is  a  3 carbon hydroxy  acid. There are two types of lactic acid: L-lactate and D-lactate. Most forms of lactic acidosis are caused by too much L-lactate.
• If lactic acid builds up in the body more quickly than it can be removed, acidity levels in bodily fluids — such as blood — spike. This buildup of acid causes an imbalance in the body’s pH level, which should always be slightly alkaline instead of acidic.
• Lactic acid buildup occurs when there’s not enough oxygen in the muscles to break down glucose and glycogen. This is called anaerobic metabolism. Elevation  of lactic  acid  in  the  circulation (normal plasma 4-1  5  mg/dl)  may  occur  due  to  its increased  production or  decreased  utilization.
• The term  oxygen  debt refers  to the  excess  amount of 0₂ required  to  recover.  In  clinical  practice, measurement  of  plasma  lactic  acid is useful  to know  about the oxygen  debt, and  monitor the patient’s  recovery

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.
• The cancer  cells get adapted  to  hypoxic glycolysis through  the involvement  of a transcription  factor namely  hypoxia-inducible  transcription  factor(HIF). HIF  increases  the synthesis  of  glycolytic enzymes  and  the  glucose  transporter.
• However, the cancer  cells  cannot  grow  and  survive  without proper  vascularization. One  of the modalities  of cancer  treatment  is  to  use  drugs  that  can inhibit vascularization  of tumors.