Transport processes

Transport processes in the aspect of metabolic engineering.

1. METABOLIC PROCESS AND ENGINEERING BY, RAJESWARI R SUJANI SATHYA KEERTHANA S JEYA CRESSIDA J

2. Transport processes

3. TRANSPORT ACROSS MEMBRANES Every living cell must acquire raw materials from its surroundings for biosynthesis and for energy production, and must release the byproducts of metabolism to its environment. Few nonpolar compounds can dissolve in the lipid bilayer and cross the membrane unassisted. But for polar or charged compounds or ions, a membrane protein is essential for transmembrane movement. In some cases a membrane protein simply facilitates the diffusion of a solute down its concentration gradient. If the transport occurs against a gradient of concentration, requires energy.

4. CONTD.., The energy may come directly from ATP hydrolysis or may be supplied in the form of movement of another solute down its electrochemical gradient with enough energy to carry another solute up its gradient.

5. TYPES OF TRANSPORT ACROSS MEMBRANES Transport process Passive transport Active transport Simple diffusion Facilitated diffusion Osmosis Primary active transport Secondary active transport

6. PASSIVE TRANSPORT In passive transport process a specific molecule flows from high concentration to low concentration . Some materials diffuse readily through the membrane, but others require specialized proteins, such as channels and transporters, to carry them into or out of the cell. A movement of biochemicals and other atomic or molecular substances across membranes that does not require an input of chemical energy.

7. PASSIVE TRANSPORT Simple diffusion Diffusion is a process of passive transport in which molecules move from an area of higher concentration to one of lower concentration. The diffusion continues till the gradient has been vanished.

8. PASSIVE TRANSPORT Facilitated diffusion Facilitated diffusion is a process by which molecules are transported across the plasma membrane with the help of membrane proteins. A concentration gradient exists that would allow ions and polar molecules to diffuse into the cell, but these materials are repelled by the hydrophobic parts of the cell membrane. Facilitated diffusion uses integral membrane proteins to move polar or charged substances across the hydrophobic regions of the membrane. 2 types of protein involved in facilitated diffusion are channel and carrier proteins.

9. PASSIVE TRANSPORT Channel protein Channel proteins can aid in the facilitated diffusion of substances by forming a hydrophilic passage through the plasma membrane through which polar and charged substances can pass. Channel proteins can be open at all times, constantly allowing a particular substance into or out of the cell, depending on the concentration gradient; or they can be gated and can only be opened by a particular biological signal.

10. PASSIVE TRANSPORT Carrier protein Another type of protein embedded in the plasma membrane is a carrier protein. This protein binds a substance and, in doing so, triggers a change of its own shape, moving the bound molecule from the outside of the cell to its interior; depending on the gradient, the material may move in the opposite direction. Carrier proteins are typically specific for a single substance. Example: Glucose transport proteins, or GLUTs, are involved in transporting glucose

11. PASSIVE TRANSPORT

12. PASSIVE TRANSPORT Osmosis Osmosis is the movement of water through a semipermeable membrane according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes. Semipermeable membranes, also termed selectively permeable membranes or partially permeable membranes. In osmosis, water always moves from an area of higher water concentration to one of lower concentration. In the diagram shown, the solute cannot pass through the selectively permeable membrane, but the water can.

13. PASSIVE TRANSPORT Osmosis

14. ACTIVE TRANSPORT Active transport is the movement of molecules across a membrane from a region of their lower concentration to a region of their higher concentration—in the direction against the concentration gradient. Active transport requires cellular energy to achieve this movement. There are two types of active transport: primary active transport that uses ATP, and secondary active transport that uses an electrochemical gradient.

15. ACTIVE TRANSPORT Primary active transport Primary active transport, also called direct active transport, directly uses energy to transport molecules across a membrane. Example: Sodium-potassium pump, which helps to maintain the cell potential.

16. ACTIVE TRANSPORT Secondary active transport Secondary active transport or co-transport, also uses energy to transport molecules across a membrane It uses the electrochemical potential difference created by pumping ions out of the cell The two main forms of secondary active transport are antiport and symport . In secondary active transport, the two molecules being transported may move either in the same direction (i.e., both into the cell), or in opposite directions (i.e., one into and one out of the cell).  

17. ACTIVE TRANSPORT CONTD.., When they move in the same direction, the protein that transports them is called a  symporter , while if they move in opposite directions, the protein is called an  antiporter .

18. ENDOCYTOSIS AND EXOCYTOSIS Endocytosis Endocytosis is the process by which cells absorb larger molecules and particles from the surrounding by engulfing them. It is used by most of the cells because large and polar molecules cannot cross the plasma membrane. Endocytosis consists of phagocytosis and pinocytosis Phagocytosis: Phagocytosis or “cell eating,”  is the process by which large particles, such as cells or relatively large particles, are taken in by a cell.

19. ENDOCYTOSIS AND EXOCYTOSIS Contd.., For example, when microorganisms invade the human body, a type of white blood cell called a neutrophil will remove the invaders through this process, surrounding and engulfing the microorganism, which is then destroyed by the neutrophil.

20. ENDOCYTOSIS AND EXOCYTOSIS Contd.., Pinocytosis: Pinocytosis, or “cell drinking,” is a process that takes in molecules, including water, which the cell needs from the extracellular fluid.

21. ENDOCYTOSIS AND EXOCYTOSIS Exocytosis: The process by which the cells direct the contents of secretory vesicles out of the cell membrane is known as exocytosis . These vesicles contain soluble proteins to be secreted to the extracellular environment, as well as membrane proteins and lipids that are sent to become components of the cell membrane.

22. FUELING REACTIONS

23. Fueling reactions serves 3 purposes Generation of Gibbs free energy in the form of ATP in, which is used to fuel other cellular reactions Production of reducing power (or reducing equivalents), in the form of cofactor NADPH required in biosynthesis reactions Formation of precursor metabolites required in the biosynthesis of building blocks The carbon and energy source are required for biosynthesis of building blocks. Some of the most frequently used substrate (act as both carbon and energy source) are sugars(Glucose, fructose, lactose, etc.) The catabolism of sugars starts with glycolysis, and the end product is pyruvate. Pyruvate is further processed in fermentative pathways, anaplerotic pathways, TCA cycle, transamination pathways for aminoacid formation.

24. Contd.., Thus many organisms have the ability to catabolize amino acids, organic acids or fats either in the presence of sugar or as sole carbon and energy source Due to the importance of fueling pathways in central carbon metabolism and biosynthesis we are going to discuss about various pathways.

25. GLYCOLYSIS

26. INTRODUCTION In glycolysis a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in ten steps, the first five of which constitute the preparatory phase The energy gain comes in the payoff phase of glycolysis (from 6 th step to 10 th step )

27. Contd..,

28. FERMENTATIVE PATHWAYS

29. 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. • O2 is not required.

30. Glycolysis Glucose + 2NAD + 2ADP 2 pyruvate + 2NADH + 2H + 2ATP +2H2O

31. Fermentation Pathways • Glycolysis is the first stage of fermentation Forms 2 pyruvate , 2 NADH, and 2 ATP • Pyruvate is converted to other molecules, but is not fully broken down to CO2 and water • Regenerates NAD+ but doesn’t produce ATP • Provides enough energy for some single-celled anaerobic species

32. Two Pathways of Fermentation • Alcoholic fermentation Pyruvate is split into acetaldehyde and CO2 Acetaldehyde receives electrons and hydrogen from NADH , forming NAD+ and ethanol • Lactate fermentation Pyruvate receives electrons and hydrogen from NADH,forming NAD+ and lactate

33. Two Pathways of Fermentation

34. Lactate Fermentation lactic acid fermentation Vigorously contracting skeletal muscle must function under lowoxygen conditions (hypoxia), NADH cannot be reoxidized to NAD B ut NAD is required as an electron acceptor for the further oxidation of pyruvate . Under these conditions pyruvate is reduced to lactate, accepting electrons from NADH and thereby regenerating the NAD necessary for glycolysis to continue.

35. CONTD.., Certain tissues and cell types (retina and erythrocytes, for example) convert glucose to lactate even under aerobic conditions. lactate is also the product of glycolysis Under anaerobic conditions in some microorganisms also .

36. Examples of fermentation pathways 1) Lactic acid fermentation Found in many bacteria; e.g . Streptococcus sp, Lactobacillus acidophilus 2) Mixed acid fermentation e.g. Escherichia coli 3) 2,3-Butanediol fermentation e.g. Enterobacter aerogenes

37. Alcohol fermentation Humans cannot ferment alcohol in their own bodies, we lack the genetic information to do so

38. Examples of alcoholic pathways 1) Ethanol e.g. ( Saccharomyces cerevesiae ) 2) B utanol e.g. ( Clostridium acetobutylicum )

39. Real Life Example Humans ferment lactic acid in muscles where oxygen becomes depleted, resulting in localized anaerobic conditions. This lactic acid causes the muscle stiffness couch-potatoes feel after beginning exercise programs. The stiffness goes away after a few days allows aerobic conditions to return to the muscle, and the lactic acid can be converted into ATP via the normal aerobic respiration pathways.

40. Fermentation Pathway as a whole

41. CITRIC ACID CYCLE AND OXIDATIVE PHOSPHORYLATION

42. INTRODUCTION Citric acid cycle is also known as TCA( tricarboxylic acid) cycle or the Krebs cycle Series of chemical reactions by aerobic organisms- generate energy through the oxidation of acetate Eukaryotic cell-mitochondria Prokaryotic cell- cytosol It is the key metabolic pathway connects carbohydrate ,fat and protein metabolism

43. CONTD.., The reactions are carried out by eight enzymes- completely oxidize acetate in the form of acetyl- CoA – 2 molecules of carbon dioxide and water. Primary source of acetyl- CoA – break down of sugars by glycolysis – pyruvate – decarboxylated by pyruvate dehydrogenase generates acetyl- CoA

44. CONTD.. , The cycle consumes acetate (in the form of acetyl- CoA ) and water, reduces NAD + to NADH, and produces carbon dioxide. The NADH generated by the TCA cycle is fed into the oxidative phosphorylation pathway. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable energy in the form of ATP.

45. BREAKDOWN OF PYRUVATE

46. CONTD .., Each pyruvate molecule loses a carboxylic group in the form of carbon dioxide. The remaining two carbons are then transferred to the enzyme CoA to produce Acetyl CoA .

47. One complete cycle gives 3 NAD+ 3NADH 1Q 1QH2 1 GDP + Pi 1GTP This NADH and QH2 are used in Oxidative phosphorylation to generate ATP

48. OXIDATIVE PHOSPHORYLATION It is the metabolic pathway – cells uses enzyme to oxidize nutrients – releases energy which is used to produce ATP.

49. COMPLEX 1 NADH dehydrogenase or complex 1 Reaction starts with binding of NADH molecule to comlplex1 and donation of 2 electrons As electrons pass through this complex four H+ are pumped from matrix to intermembrane space.

50. COMPLEX 11 Succinate dehydrogenase or complx 11 It oxidizes succinate to fumerate and reduces ubiquinone As this reaction releases less energy than oxidation of NADH, it does not transport protons across membrane .

51. COMPLEX III Cytochrome c reductase or complex III Cytochrome is a kind of electron transfering protein contains atleast one heme group. COMPLEX IV This mediates final reaction in electron transport chain and transfers electros to oxygen.

52. COMPLEX V ATP synthase uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP, ADP and Pi . NADH 3ATP SUCCINATE 2ATP

53. THANK YOU