The following points highlight the five major pathways in organisms. The pathways are: 1. Glycolysis 2. Pentose Phosphate Pathway 3. Entner-Doudoroff Pathway 4. Tricarboxylic Acid Cycle 5. Glyoxylate Cycle.
Metabolic Pathway # 1. Glycolysis:
Glycolysis (glyco-sugar of sweet, lysis-breakdown) is the initial phase of metabolism during which the organic molecule glucose and other sugar are partially oxidized to smaller molecules e.g. pyruvate usually with the generation of some ATP and reduced coenzymes. Microorganisms employ several metabolic pathways to catabolize glucose and other sugars.
There are three important routes of glucose conversion to pyruvate such as glycolysis or Embden-Myerhof pathway (BMP) pathway, pentose phosphate pathway, and Entner-Doudroff pathway. Glycolysis is the most important type of mechanism by which organisms obtain energy from organic compounds in absence of molecular oxygen. As it occurs in the absence of oxygen, therefore, it is also called anaerobic fermentation.
Since living organisms arose in the environment lacking oxygen, anaerobic fermentation was the only method to obtain energy. However, glycolysis or anaerobic fermentation is present in both aerobic and anaerobic organisms.
Most higher organisms have retained the glycolytic pathway of degradation i.e. glucose to pyruvic acid as a preparatory pathway for complete aerobic catabolism of glucose. Glycolysis also serves as an emergency mechanism in anaerobic organisms to produce energy in the absence of oxygen.
(i) EMP Pathways:
In case of aerobic catabolic carbohydrate metabolism (aerobic respiration), some bacteria such as E. coli, Azotobacter spp., Bacillus eutrophus, etc. exhibit EMP pathway whereas, ED pathway (phosphorylated) is followed by the species of Alcaligenes, Rhizobium, Xanthomonas, etc. The non-phosphorylated ED pathway occurs in archaea (Pyrococcus spp., Thermoplasma spp, etc.). It is interesting to note that no archaeobacteria uses EMP pathway.
EMP pathway in bacteria initiates by using the phosphoenol pyruvate phosphotransferase system (PEP: PTS) that converts glucose to glucose 6-phosphate during nutrient transport across the cell membrane.
The glucose 6-phosphate is then isomerized to fructose 6-phosphate which is further converted to fructose 1, 6-bi-phosphate. This conversion requires ATP as a source of energy and an enzyme called phosphofructokinase.
It is essentially the reversal of glycolysis, which fulfills a similar anaplerotic role. It is particularly important during growth on pyruvate related C3 compounds and C2 units. The several class of flow of carbon from pyruvate maintains a supply of hexoses. These are required for cell wall and its component synthesis.
The complete pathway of glycolysis from glucose to pyruvate (Fig. 12.4) were elucidated by Gustav Embden (who gave the manner of cleavage of fructose 1, 6-diphosphate and pattern of subsequent steps) and Otto Meyerhof (who confirmed Embden’s work and studied the energetics of glycolysis), in late 1953s. Therefore the sequence reaction from glucose to pyruvate is also called Embden-Meyerhof pathway or glycolysis (EMP).
The overall balance sheet of glycolysis is given below:
Glucose + 2ADP + 2Pi + 2NAD+ → Pyruvate + 2ATP + 2NADH + 2H+
In anaerobic organisms pyruvate is further converted to lactate or other organic compounds like alcohol, etc., after using NADH and H+ formed during glycolysis:
Pyruvate + NADH + H+ ↔ Lactate + NAD+
In aerobes the pyruvate is converted to acetyl CoA as a preparatory step for entrance into tricarboxylic acid cycle, for complete oxidation of glucose.
Pyruvate + NAD+ + CoA → Acetyl CoA + NADH + H+ + CO2
Glycolysis is carried out by the help of ten enzymes for ten reactions of the glycolytic pathway. These enzymes are present in the soluble portion of the cytoplasm. All the mtermediates of the glycolytic pathway are phosphorylated compounds. The most important use of phosphate groups is in the production of ATP from ADP and phosphate.
The complete reactions of glycolytic pathway can be divided into two stages. In the first stage, ATP is utilized and glucose is converted into two molecules of three carbon compounds, glyceraldehyde 3-phosphate and dihydroxy acetone phosphate. The glyceraldehyde 3-phosphate is converted into pyruvic acid resulting in a net synthesis of two molecules of ATP.
The complete reaction with respective enzyme is shown in Fig. 12.4:
Apart from glucose, other types of sugar (monosaccharides, disaccharides, polysaccharides) can also enter the glycolytic pathway.
(а) Polysaccharides e.g. Glycogen:
Glucose-6-phosphate Glucose 6 – phosphate can enter as an intermediate of glycolysis.
(b) Disaccharides e.g. Sucrose:
The three key regulatory enzymes, hexokinase, phosphofructokinase and pyruvate kinase act irreversibly and rest of the steps are reversible.
(c) Homo-saccharides: e.g. Fructose:
Fructose can enter the glycolysis by changing to glyceraldehyde 3-phosphate.
Dihydroxyacetone phosphate can enter the glycolysis after enzymatically converting to dihydroxyacetone phosphate.
(ii) Alternate EMP Pathway-Methyl Glyoxal Pathway:
The methyl glyoxal pathway is an alternate of the EMP pathway. It Operates in the presence of low concentration of phosphate to the bacteria, E. coli, Clostridium spp., Pseudomonas spp. etc. In this pathway, dihydroxyacetone so formed converted to methyl glyoxal which later on gives rise to pyruvate.
Hence, there is complete absence of the phosphorylation step in which glyceraldehyde 3-phosphate forms 1, 3-bis-phospho- glycerate. The methyl glyoxal pathway consumes O2 and ATP and no ATP is generated in this pathway (Fig. 12.5).
Metabolic Pathway # 2. Pentose Phosphate Pathway (PPP):
Pentose phosphate pathway is an alternative of glucose degradation. This pathway, also called hexose monophosphate shunt (HMP) or phosphogluconate pathway is not the major pathway, but is a multipurpose pathway. Its main function is to generate reducing power in the extra-mitochondrial cytoplasm in the form of NADH. Its second function is to convert hexoses into pentoses, required in synthesis of nucleic acids.
Its third function is complete oxidative degradation of pentose. The reactions of phosphogluconate pathway take place in the soluble portion of extra-mitochondrial cytoplasm of cells.
The bacteria which show PPP are Bacillus subtilis, E. coli. Streptococcus faecalis and Leuconostoc mesenteroides. Apart from microorganisms the prominent tissues which show PPP are liver, mammary gland and adrenal cortex. The complete PPP is given in Fig 12.6.
There are three enzymes involved in PPP i.e. transketolase, transaldolase and ribulosephosphate 3-epimerase. Ribulose phosphate 3-epimerase catalyzes the conversion of ribulose 5-phosphate into the epimer xylulose 5-phosphate. Transketolase transfers the glycoaldehyde group (CH, OH—CO—) from xylulose 5-phosphate to ribose 5-phosphate to yield sedoheptulose 7-phosphate and glyceraldehyde-3-phosphate.
Transketolase also catalyzes the transfer of glycoaldehyde group from a number of 2-keto sugar phosphate to carbon atom one of a number of different aldose phosphate. Transaldolase acts on the products of transketolase and transfer dihydroxyacetone group to form fructose 6-phosphate and erythrose 4-phosphate (Fig. 12.6).
Fig. 12.6 : The Pentose phosphate pathway.
Pentose phosphate pathway thus functions according to the needs of the cell. If the requirement of reducing power is more then it proceeds towards the formation of NADPH but if pentoses are required it functions in the direction of formation of pentose. But if the cell requires instant energy the PPP stops and glycolysis and TCA proceed.
(a) To anabolic reactions that require electron donors
(b) To Calvin-Benson Cycle (dark reactions of photosynthesis)
(c) To synthesis of nucleotides and nucleic acids
(d) To step e of glycolysis
(e) To glucose 6-phosphate which can enter the pentose phosphate pathway or glycolysis
(f) To synthesis of several amino acids.
Metabolic Pathway # 3. Entner-Doudoroff Pathway:
Apart from glycolysis, Entner-Doudoroff pathway is another pathway for oxidation of glucose to pyruvic acid. This pathway is found in some Gram-negative bacteria like Rhizobium, Agrobacterium and Pseudomonas and is absent in Gram-positive bacteria. In this pathway each molecule of glucose, forms two molecules of NADPH and one molecule of ATP. The complete pathway is shown in the Fig. 12.7.
In this pathway glucose 6-phosphate is oxidized to 6-phosphogluconate, then converted to 2- keto-3-deoxy-6-phosphoglucose (kDPG) cleaved using enzyme to give rise glyceraldehyde’s 3- phosphate and pyruvate directly without generation of ATP. The catabolism of glucose results in production of only one ATP molecule whereas in EMP pathway, two ATP molecules are produced. This seems that EMP pathway more efficient than that of ED pathway.
Further, difference between ED pathway and PP pathway is the generation of reduced NADPH from NADP in the former. It is interesting to note that coenzyme NADP+ and NADPH are used in anabolic reactions. Thus, the ED pathway provides an important mechanism for producing NADPH and the 3-carbon building blocks used in biosynthetic reactions etc.
Partially non-phosphorylated ED pathway is involved in some bacteria such as Clostridium spp. Achromobacter spp., Alcaligens spp. and Archaea (Halobacterium spp.) In this case, intermediate product formed prior to kDPG is non-phosphorylated, and phosphogluconate is dehydrated to give rise kDPG, which gives to pyruvate.
In later steps, the reactions of ED pathway are followed. This pathway is also found in other bacteria such as Pseudomonas aeruginosa, Azotobacter, and Enterococcus faecalis, and an anaerobic bacterium Zymomonas mobilis.
Metabolic Pathway # 4. Tricarboxylic Acid Cycle:
The tricarboxylic acid cycle was first given by H.A. Krebs in 1937. H.A. Krebs then gave the name citric acid cycle. Because of citric acid is the first product of Krebs cycle, is also known as TCA cycle due to presence of three carboxylic groups in a molecule of citric acid.
The cycle is of universal occurrence in all the aerobic organisms and leads to complete oxidation of glucose to CO2 and H2O while glycolysis leads to incomplete oxidation of glucose to pyruvate.
Tri-carboxyhc acid cycle completely oxidises it to release large amount of energy in the form of NADH + H+ mainly and GTP. NADH + H+ enter the respiratory chain where each NADH + H+ produces three ATP molecules. GTP is converted to ATP by substrate level oxidation. Another form of energy is in the form of substrate of FADH2, which also enters the respiratory chain to form two molecules of ATP.
All the reactions of tricarboxylic acid cycle take place in the inner compartment of mitochondrion. Some of these enzymes occur in the matrix of inner compartment, while rest of them occur on the inner mitochondrial membrane. For the start of the cycle, the pyruvate formed in the glycolysis is first converted to acetyl Co-A by preparatory reaction.
Pyruvate + NAD+ + CoA → acetyl CoA + NADH + H+ + CO2
The reaction is irreversible and is not itself a part of the tricarboxylic acid cycle. It is carried out with the help of the enzyme pyruvate dehydrogenase. Acetyl CoA then enters the cycle after combining with oxaloacetate to form citrate, after which a cycle of reactions occurs (Fig. 12.8) leading to the formation of six CO2, eight NADH + H+, one FADH2 and one molecule of glucose.
There are few key steps in the tricarboxylic acid cycle which control the cycle as per need of the cell. The first of these controls is the preparatory reaction. The activity of pyruvate dehydrogenase is reduced in the presence of excess ATP and again increases in the absence of ATP.
There are two more steps which can control the cycle. These are the isocitrate dehydrogenase reaction (which requires ADP as positive regulation), and succinate dehydrogenase reaction (promoted by succinate, phosphate and ATP). However, the key control of the cycle is the reaction carried out by citrate synthase. This is the primary control of the cycle.
Metabolic Pathway # 5. Glyoxylate Cycle:
It is anaplerotic reaction in which oxaloacetate is taken from TCA cycle to meet out the demand of carbon requirement for amino acid biosynthesis. Hence, these intermediates have to be replenished via an alternate route, called anaplerotic pathway i.e. glyoxylate pathway. This cycle operates for glucoiieogenesis. Glyoxylate cycle was given first by Krebs and H.R. Kornberg.
This cycle is a modified form of tricarboxylic acid cycle found in plants and those microorganisms which utilize fatty acids as the source of energy in the form of acetyl Co A.
In this cycle the CO2 evolving steps of tricarboxylic acid cycle were by-passed and instead a second molecule of acetyl CoA is utilized (which condenses with glyoxylate to form malate). Succinate is a by product, used for biosynthesis, particularly in gluconeogenesis.
The overall reaction of glyoxylate cycle is given below:
2 Acetyl Co-A + NAD+ + 2H2O → Succinate + 2CoA + NADH + H+
The two key enzymes, isocitrate lyase and malate synthase of glyoxylate cycle are localised in cytoplasmic organelles called glyoxysomes. Glyoxylate cycle goes on simultaneously with the tricarboxylic acid cycle, while tricarboxylic acid provides energy; glyoxylate cycle provides succinate for the formation of new carbohydrate from fats as shown in Fig. 12.9.