04 Metabolism Revised-2 (1).pptx aau baca

microbial metabolism, catabolism, anabolism, microbes, glycolysis, etc , krebs cycle ...

1. Microbial Metabolism Central Role of Glucose

2. Metabolism pathways

3. Metabolism Includes all the chemical processes within cells and tissue that are concerned with their building up and breaking down and their functional operations. Energy Metabolism is energy composition of metabolism and deals with the overall energy production and requirement of the organisms. Anabolism : Process union of smaller into larger molecules or metabolism of tissue formation. Catabolism : process of breakdown primarily concerned with the splitting of the larger protoplasmic molecules into the smaller ones. ETC: Electron transport chain NADH: Nicotinamide adenine dinucleotide hydrogen NADPH: Nicotinamide adenine dinucleotide phosphate hydrogen Terms regarding Metabolism

4. Most organisms metabolize glucose though the EMP or Glycolysis ( Embden Meyerhof- Parnas ) pathway to generate ATP, pyruvate and NADH. The HMP or PP pathway is needed to supply the metabolic intermediates not available from the EMP pathway such as pentose-5-phosphate and erythrose-4-phosphate, and NAPDH . Some prokaryotes metabolize glucose through unique pathways known only in prokaryotes, e.g. the Entner-Doudoroff (ED) pathway and phosphoketolase (PK) pathway . Some prokaryotes have genes for the ED pathway in addition to the EMP pathway . Carbohydrates are phosphorylated before they are metabolized in most cases . It is believed that phosphorylated intermediates are less likely to diffuse away through the cytoplasmic membrane. Introduction to Metabolism

5. Carbohydrate metabolism in the animal body is essentially the metabolism of glucose and of the substances related to glucose in their metabolic processes. Glucose occupies a central position in the metabolism of plant, animals and may microbes. Glycolysis and Krebs cycle synthesis 10% of ATP. Electron transport chain synthesis 90% of ATP. Fate of Pyruvate

6. Glycolysis It is a pathway first described by scientist Embden , Meyerhoff and Parnas in 1940 so also called as EMP pathway It is the sequence of reactions converting glucose to pyruvate or lactate, with production of ATP It takes place in all cells of body, enzymes of this pathway are present in cytoplasm of cell. It is operated in both aerobic and anaerobic condition. It is major pathway for ATP synthesis in tissues lacking mitochondria, e.g. erythrocytes, cornea, lens etc. Very essential for brain – depend on glucose for energy. Participated in both catabolism and anabolism of glucose So , also called as Amphibolic pathway

7. Almost universal central pathway of glucose catabolism. For glycolysis two ATP and two NADH molecules are generated. Two phases Primary Phase Secondary phase Glycolysis /EMP( Embden -Meyerhof glycolytic Pathway)

8. Primary phase Glycolysis converts glucose to pyruvate . -a 10-step biochemical pathway -occurs in the cytoplasm -2 molecules of pyruvate are formed -net production of 2 ATP molecules by substrate-level phosphorylation -2 NADH produced by the reduction of NAD + …cont Glucose is phosphorylated twice by adding 2 P coming from 2 ATP ( substrate-level- phosphorylation ). Thus, Glucose (6- C ) splits into two small sugar molecules (each with 3- C ).

9. Secondary (Payoff) phase ATP is produced by substrate-level phosphorylation and NAD + is reduced to NAD H . 4 ATP and 2NADH are produced per glucose. Thus, the net yield from glycolysis is 2 ATP and 2 NADH per glucose. Oxygen is not required for glycolysis

10. Phosphorylation of glucose- Required enzyme hexokinase and a phosphate of ATP is transferred to glucose on its 6 th carbon. 1.Co-factor is mg + , na + , K + . 2. Reaction is irreversible. 3.Present in all cells. 4. Have low k m value. Glucokinase present in liver, have high k m value. Isomerization of glucose-6- phosphate- Enzyme hexoseisomerase act on glucose -6- phosphate and convert it in to fructose-6-phosphate. Reaction is stereospecific ,reversible and requiress mg +. Formation of fructose 1,6 diphosphate -  Fructose-6-phosphate is converted into fructose 1,6 diphosphate by the enzyme phosphofructokinase . The reaction is irreversible and phosphofructokinase enzyme is allosteric and step is regulatory step in Gycolysis. 1.Energy investment phase

11. CLEAVAGE OF FRUCTOSE 1,6 DIPHOSPHATE Enzyme aldolase cleave the fructose 1,6 diphosphate to form two molecules each contain 3 carbon , glyceraldehyde 3-phosphate and dihydroxy acetone phosphate( DHAP). 2. splitting phase CONVERSION OF DHAP TO GLYCERALDEHYDE 3-PHOSPHATE   DHAP is converted into glyceraldehyde 3-phosphate and enzyme involved is Trioseisomerase . Reaction is stereospecific and reversible. Thus 2 molecules of glyceraldehyde 3-phosphate are obtained from one molecule of glucose .

12. Oxydation of 3- PGAL- Only step in glycolysis - oxidation occur.-CHO group of 3- PGAL is oxidized to form carboxylic group.Enzyme required- glyceraldehyde 3-phosphate dehydrogenase . imp step involved in the formation of NADH+ h + and high energy compond 1,3-bisphospho-glycerate. Arsenite and iodoacetate inhibit reaction. Formation of 3- phosphoglyceric acid (PGA)- 1,3 di PGA is converted to 3-PGA. Enzyme is 3- PGA kinase . Phosphate is transferred to ADP and form ATP. Good e.g. Of substrate level phosphorylation , since ATP synthesized from the substrate without the involvement of electron transport chain. Energy generation phase

13. Formation of 2- PGA Enzyme phosphoglycerate mutase catalyse the reversible shift of phosphate group between C2 and C3 caron of glyceric acid. High energy phosphate on 3 rd carbon transferred to 2 nd carbon of 3-PGA. Mg2+ is essential for this reaction. Formation of phospho enol pyruvate ( PEP ) 2- PGA is converted to PEP by enzyme enolase . Reaction is stereospecific and reversible . Here, Enzyme enolase require Mg2+ ions . Fluoride can inhibit this reaction. Formation of pyruvic acid   Substrate level phosphorylation occur. High energy phosphate to ADP and form ATP and Pyruvic acid

14. Summary of Glycolysis , ATP , ATP {Glucose g G6P g F6P g F1,6BP g DHAP g GAP } a Preparatory Phase ; NADH ; ATP ; ATP {GAP g 1,3BPG g 3PG g 2PG g PEP g Pyruvate } a Payoff Phase The net gain is of two molecules of ATP and two molecules of NADH in the process of glycolysis .

15. 1.Glycolysis play a key role in fuelling metabolism of cell by generating reducing power. 2. ATP and some metabolic precursor metabolites formed are hexose phosphate, 3-PGA, PEP and Pyruvic acid. 3 . During operation of glycolysis reduced NAD is formed which can serve as potential reducing power. 4. The pathway also provide possibly of ATP generation by both substrate level phosphorylation and oxidative phosphorylation . SIGNIFICANCE OF GLYCOLYSIS The overall process of glycolysis is: Glucose + 2 NAD + + 2 ADP + 2 P i → 2 Pyruvate + 2 NADH + 2 H + + 2 ATP + 2 H 2 O

16. Entner-Doudoroff (ED) pathway Glycolytic pathways in some G(-) bacteria The ED pathway is an unique glycolytic pathway in prokaryotes. The ED pathway was first identified in Pseudomonas saccharophila by Entner and Doudoroff . The ED pathway turned out to be the main glycolytic pathway in prokaryotes that do not possess enzymes of the EMP pathway. The ED pathway functions as the main glycolytic pathway in other G(-) bacteria such as Zymomonas species.

17. Entner-Doudoroff (ED) pathway Glycolytic pathways in some G(-) bacteria (continued)

18. Entner-Doudoroff (ED) pathway Key enzymes of the ED pathway NAD(P) + -dependent glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to 6-phosphogluconolactone that is converted to 6-phosphogluconate. 6-Phosphogluconate dehydratase removes water molecule from 6-phosphogluconate and produces 2-keto-3-deoxy-phosphogluconate (KDPG). KDPG aldolases splits KDPG into pyruvate and glyceraldehyde-3-phosphate. The key enzymes in the ED pathway are 6-phosphogluconate dehydrogenase and KDPG aldolase.

19. Entner-Doudoroff (ED) pathway Key enzymes of the ED pathway (continued) The ED pathway has a net yield of 1 ATP for every glucose molecules as well as 1 NADH and 1 NADPH. In comparison, glycolysis has a net yield of 2ATP and 2 NADH for one glucose molecule. On Wikipedia.com

20. Entner-Doudoroff (ED) pathway Modified ED pathways Unusually, some prokaryotes oxidize glucose and the intermediates are phosphorylated before being metabolized in a similar manner as in the ED pathway. On Wikipedia.com Extracellular oxidation of glucose by G(-) bacteria

21. Entner-Doudoroff (ED) pathway Extracellular oxidation of glucose by G(-) bacteria Some strains of Pseudomonas oxidize glucose extracellularly when the glucose concentration is high. These bacteria possess glucose dehydrogenase and gluconate dehydrogenase on the periplasmic face of the cytoplasmic membrane. When glucose is depleted, gluconate and 2-ketogluconate are transported through specific transporters and phosphorylated , consuming ATP. 2-Keto-6-phosphogluconate is reduced to 6-phosphogluconate by a NADPH-dependent reductase . Gluconate and 2-ketogluconate are uncommon in nature, and few microbes use these compounds. The ability to oxidize glucose and to use its products might therefore be advantageous for those organisms capable of doing this.

22. Entner-Doudoroff (ED) pathway Modified ED pathway in archaea Hyperthermophilic archaea ( Sulfolobus , Thermoplasma , T hermoproteus ) metabolize glucose to pyruvate and glyceraldehyde without phosphorylation in a similar way as the ED pathway. A halophilic archaeon , Halobacterium saccharovorum oxidize glucose to 2-keto-3-deoxygluconate. The archaeal glucose dehydrogenase is a NAD(P) + -dependent enzyme.

23. The citric acid cycle was proposed in 1937 by Hans Krebs . Involvement of several metabolites in cellular oxidative processes was recognized. In 1935, Albert Szent & Gyorgyi demonstrated that cellular respiration is accelerated by catalytic amounts of succinate , fumarate , malate , or oxaloacetate . Interconverted sequence: Citrate , cis-aconitate , isocitrate, α - ketoglutarate , succinate , fumarate , malate , oxaloacetate etc In 1936, Martius and Knoop demonstrated that citrate formed from oxaloacetate & pyruvate by H 2 O 2 under basic condition. Idea was caught by Kreb and he completed the cycle. TCA CYCLE

24. TCA involves the oxidation of acetyl coA to CO 2 and H 2 O. Utilizes about 2/3 total oxygen consumed by body. TCA occurs in mitochondria in eukaryotes and in bacteria- cytosol. About 65-70% of ATP is synthesized in Krebs cycle. Name TCA because three tricarboxylic acids – citrate, cis-aconitate, and isocitrate participates It is most imp central pathway connecting almost all individual metabolic pathways and amphibolic in nature. Oxaloacetate plays a catalytic role in TCA cycle. Key part of aerobic respiration in cells. This cycle is also called the Krebs cycle and the citric acid cycle. Silent features of TCA cycle

25. Intermediatery step between Glycolysis and TCA cycle 26 1 st decarboxylation

26. This shows the TCA cycle in the context of what is happening in the inner mitochondrial membrane. It's role can be seen as giving energy to the reduced coenzyme NADH which then powers the ETC in the membrane . The energy is used by the protein complex to produce a proton gradient which in turn powers ATP synthase in its role of producing the needed ATP

27. ENERGETICS

28. Kreb’s cycle generates 2 CO 2 molecules, 3 NADH, one FADH 2 & 1 GTP molecules . For each acetyl CoA molecule oxidized, the generation of ATP from NADH & FADH 2 molecules associated with electron transport chain & oxidative phosphorylation . The generation of GTP molecule takes place via substrate level phosphorylation . Acetyl- CoA + 3 NAD + FAD + GDP + Pi + 2H 2 O → 2CO 2 + 3 NADH + FADH 2 + GTP + 2H + + CoA

29. Function NADPH production Reducing power carrier Synthetic pathways Role as cellular antioxidants Ribose synthesis Nucleic acids and nucleotides Overview of PP/HMP pathway

30. Hexose monophosphate (HMP) pathway The HMP pathway is also called the pentose phosphate (PP) pathway. When Escherichia coli grows on glucose as the sole carbon source, the EMP and HMP pathways consume 72% and 28% glucose, respectively, indicating that the EMP cannot meet all the requirements for biosynthesis. The HMP pathway provides some precursors (pentose-5-phosphate and erythrose-4-phosphate) and a reducing power (NADPH) to the biosynthetic metabolism. When glucose is only metabolized through the EMP pathway and TCA cycle, NADPH is produced only by isocitrate dehydrogenase (Section 5.2). NADH is seldom used in biosynthetic reactions. Most of the NADPH needed for biosynthesis arises from the HMP pathway.

31. HMP/PP pathway The pentose phosphate pathway is an alternate route for the oxidation of glucose where ATP (energy) is neither produced nor utilized. The Pentose phosphate pathway takes place entirely within the cytoplasm(because NADP + is used as a hydrogen acceptor) and is also known as the hexose monophosphate shunt or phosphogluconate pathway. Is basically used for the synthesis of NADPH and D- ribose

32. Hexose monophosphate (HMP) pathway HMP pathway in three steps

33. Hexose monophosphate (HMP) pathway In addition to supplying precursors and reducing powers, the HMP pathway is the major glycolytic metabolism in microbes that (1) utilize pentoses , (2) do not possess other glycolytic activities The HMP cycle is also employed for the complete oxidation of sugars in bacteria lacking a functional TCA cycle. Additional functions of the HMP pathway In addition to supplying precursors and reducing powers, the HMP pathway is the major glycolytic metabolism in microbes that (1) utilize pentoses , (2) do not possess other glycolytic activities The HMP cycle is also employed for the complete oxidation of sugars in bacteria lacking a functional TCA cycle. Additional functions of the HMP pathway

34. Hexose monophosphate (HMP) pathway Pentoses such as xylose and arabinose are phosphorylated and metabolized to fructose-6-phosphate and glyceraldehyde-3-phosphate through steps 2 and 3 of the HMP pathway. Utilization of pentoses Thiobacillus novellus and Brucella abortus oxidize glucose completely although they lack a functional EMP or ED pathway. Glucose is oxidized through the oxidative HMP cycle. Oxidative HMP cycle

35. Regulation of Hexose monophosphate (HMP) pathway Regulation of the HMP pathway is exerted through control of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase , Which are inhibited by the accumulation of NADPH and NADH . In the HMP and ED pathways, dehydrogenation of glucose-6-phosphate is a common reaction, but is catalyzed by separate enzymes . The NADP + -dependent HMP pathway enzyme is inhibited by NAD(P)H but not by ATP . The enzyme in the ED pathway uses NAD + as the electron acceptor and is inhibited by ATP and PEP.

36. Electron transport chain ( oxidative phosphorylation ) When one glucose molecule is oxidized to 6 CO 2 molecules by way of glycolysis & TCA cycle , only the equivalent of 4 ATP molecules is directly synthesized Most of the ATP molecules are generated from the oxidation of NADH , the reduced form of nicotinamide dinucleotide (NAD + ) in the electron transport chain . In prokaryotic microorganism (bacteria) ETC operates in plasma membrane.

37. Electron transport chain of E.coli transports electrons from NADH (NADH is the electron donor) to acceptor & move protons (H + ) across the plasma membrane. E.coli transport chain is short, it consists of 2 branches ( cytochrome d branch & cytochrome O branch) & different cytochromes (e.g. cyt b 558 , cyt b 562 , cyt d, cyt o). Coenzyme Q ( ubiquinone ) carries electrons & donates them to both branches but the branches operate under different growth conditions.

38. RESPIRATION The two types of respiration are distinguished by the presence or absence of oxygen Not all living things need oxygen to survive and even those who do can often continue cellular activities with small amounts of oxygen present.

39. AEROBIC RESPIRATION Aerobic respiration occurs in the presence of oxygen. The products of aerobic respiration are carbon dioxide, water and ATP. One glucose molecule (C 6 H 12 O 6 ) can produce 36 ATP molecules. The following formula summarizes the reactions involved in aerobic respiration: C 6 H 12 O 6 + 6O 2 + 36 ADP + 36Pi ͢ 6CO 2 + 6H 2 O + 36 ATP

40. AEROBIC RESPIRATION During aerobic respiration, a larger number of hydrogen atoms are removed from glucose molecules than in anaerobic respiration. Every time a hydrogen atom is released an electron enters the electron transport chain. This allows aerobic respiration to produce more energy in the form of ATP than anaerobic respiration.

41. AEROBIC RESPIRATION Oxygen is so important to this process because it is the final acceptor in the electron transport chain; without it the process would stop.

42. ANAEROBIC RESPIRATION Anaerobic respiration takes place in the absence of oxygen . Anaerobic respiration is an alternate mode of energy generation in which an exogenous electron acceptor other than O 2 is used in electron transport chain leading to a proton motive force . The electron acceptors used in anaerobic respiration include nitrate (NO 3- ), sulphate (SO 4 -2 ), Carbonate (CO 2 ), Ferric ion(Fe +3 ) & even certain organic compounds.

43. TYPES OF ANAEROBIC RESPIRATION Nitrate (NO -3 ) respiration, Sulphate (SO 4 2- ) respiration, Lactic acid fermentation and Alcoholic fermentation. Lactic acid fermentation occurs in the cells of animals, in particular in the hardworking muscle cells. Glucose is partially broken down into lactic acid and energy is released in the process.

44. Types of anaerobic respiration 1 . NITRATE (NO -3 ) RESPIRATION (Nitrate Reduction):- Nitrate (NO -3 ) is one of the common type of inorganic electron acceptor used in anaerobic respiration & is reduced to NO 2- , NO, N 2 O & N 2 . The products of nitrate respiration are all gaseous, they can be easily released to atmosphere & because of this process is called denitrification . 2. SULPHATE (SO 4 2- ) RESPIRATION:- The end product of sulphate respiration is H 2 S an important natural product that participates in many biogeochemical processes.

45. Fermentation Features of fermentation pathways Pyruvic acid is reduced to form reduced organic acids or alcohols. The final electron acceptor is a reduced derivative of pyruvic acid NADH is oxidized to form NAD: Essential for continued operation of the glycolytic pathways. O 2 is not required. No additional ATP are made. Gasses (CO 2 and/or H 2 ) may be released

46. Lactic Acid Fermentation During strenuous exercise lactic acid can accumulate in the muscles when insufficient amounts of oxygen are being delivered to the muscles . An accumulation of lactic acid causes pain and cramping in the area which will increase and intensify if exercise continues.

47. Alcoholic Fermentation Alcoholic fermentation occurs in the cytoplasm of yeast cells. Alcoholic fermentation also only produces 2 molecules of ATP. During alcoholic fermentation an enzyme is used to break down glucose molecules and the result is carbon dioxide (CO 2 ) and alcohol (C 2 H 5 OH ). Champagne bubbles are results of the release of CO 2 gas.

48. Fermentation Glycosis : Glucose ----->2 Pyruvate + 2ATP + 2NADH Fermentation pathways a. Homolactic acid F. P.A -----> Lactic Acid eg . Streptococci, Lactobacilli b. Alcoholic F. P.A -----> Ethyl alcohol eg . yeast

49. Biosynthesis of Macromolecules

50. Lipid Biosynthesis Fatty acid biosynthesis- Acetyl- CoA fatty acid (cell structure) Poly-ß- hydroxybutyric acid:Acetyl-CoA PHB (storage) Phospholipid - Glycolytic intermediate lipid (membrane)

51. Amino Acid Biosynthesis Amination – addition of an amine group (N containing) to a critical intermediate Transamination - new amino acids are made from the amine group from old amino acids

52. Nucleotide Biosynthesis N molecule (amino acid ),five carbon sugar (Ribose/ Deoxyribose ), phosphate combine and synthesize nucleotides (DNA, RNA) Nitrogen base Pryrimidines - cytosine(C), thymine(T) Purines - adenine(A), guanine(G)

53. Polysaccharide Biosynthesis Peptidoglycan - Glycolytic intermediates, nucleotides forms PEG Lipopolysaccharide - Glycolytic intermediates , other sugars forms LPS , teichoic acid, mycolic acid, glycogen, etc.

54. Nutrition

55. Energy /carbon classes of organisms