Cellular physiology

1. Cellular physiology ATP and Biological Energy

2. The complexity of metabolism This schematic diagram traces only a few hundred of the thousands of metabolic reactions that occur in a cell. The dots represent molecules, and the lines represent the chemical reactions that transform them. The reactions proceed in stepwise sequences called metabolic pathways, each step catalyzed by a specific enzyme.

3. Metabolism, Energy and Life • The chemistry of life is organized into metabolic pathways • Metabolism - totality of an organism’s chemical processes • Metabolic reactions are organized into pathways that are orderly series of enzymatically controlled reactions.

4. Metabolism, Energy and Life Metabolic pathways are generally of two types: Catabolic pathways – release energy by breaking down complex molecules to simpler compounds (cellular respiration which degrades glucose to carbon dioxide and water; provides energy for cellular work); Anabolic pathways – consume energy to build complicated molecules from simpler ones (photosynthesis which synthesizes glucose from CO2 and H2O; any synthesis of a macromolecule from its monomers).

5. Organisms transform energy • Energy is a capacity to do work • Kinetic energy – energy in the process of doing work (energy of motion). • Heat (thermal energy) is kinetic energy expressed in random movement molecules. • Potential energy – energy that matter possesses because of its location or arrangement. • In the earth’s gravitational field, an object on a hill or water behind a dam have potential energy. • Chemical energy is potential energy stored in molecules because of the arrangement of nuclei and electrons in its atoms

6. Organisms transform energy • Energy can be transformed from one form to another • Kinetic energy of sunlight can be transformed into the potential energy of chemical bonds during photosynthesis. • Potential energy in the chemical bonds of gasoline can be transformed into kinetic mechanical energy which pushes the pistons of an engine

7. The energy transformations of life are subject to two laws of thermodynamics Thermodynamics – study of energy transformations First Law of Thermodynamics – energy can be transferred and transformed, but it cannot be created or destroyed (energy of the universe is constant) Second Law of Thermodynamics – every energy transfer or transformation makes the universe more disordered (every process increases the entropy of the universe).

8. The energy transformations of life are subject to two laws of thermodynamics Entropy – quantitative measure of disorder that is proportional to randomness Closed system – collection of matter under study which is isolated from its surroundings Open system – system in which energy can be transferred between the system and its surroundings.

9. The energy transformations of life are subject to two laws of thermodynamics • The entropy of a system may decrease, but the entropy of the system plus its surroundings must always increase. • Highly ordered living organisms do not violate the second law because they are open systems. • For example, animals: • Maintain highly ordered structure at the expense of increased entropy of their surroundings. • Take in complex high energy molecules as food and extract chemical energy to create and maintain order. • Return to the surroundings simpler low energy molecules (CO2 and water) and heat.

10. The complexity of metabolism • An unstable system is rich in free energy. • It has a tendency to change spontaneously to a more stable state. • In this case, free energy is proportional to the girl's altitude. • The free-energy concept also applies on the molecular scale, in this case to the physical movement of molecules known as diffusion.

11. The complexity of metabolism (c) Chemical reactions also involve free energy. The sugar molecule on top is less stable than the simpler molecules below. When catabolic pathways break down complex organic molecules, a cell can harness the free energy stored in the molecules to perform work.

12. Organisms live at the expense of free energy • Free energy: a criterion for spontaneous change • It is the amount of energy that is available to do work. • Free energy (G) is related to the system’s total energy (H) and its entropy (S) in the following way: • G= H – TS • Where • G = Gibbs free energy • H = enthalpy of total energy • T = temperature in K • S = entropy • Free energy (G) = Portion of a system’s energy available to do work; is the difference between the total energy (enthalpy) and the energy not available for doing work (TS).

13. Organisms live at the expense of free energy • The maximum amount of usable energy that can be harvested from a particular reaction is the system’s free energy change from the initial to the final state. • This change in free energy (ΔG) is given by the Gibbs-Helmholtz equation at constant temperature and pressure: • ΔG = ΔH – TΔS • Where: • ΔG = change in free energy • ΔH = change in total energy (enthalpy) • ΔS = change in entropy • T = absolute temperature in K (C+273)

14. Free energy and equilibrium • There is a relationship between chemical equilibrium and the free energy change (ΔG) of a reaction: • As a reaction approaches equilibrium, the free energy of the system decreases (spontaneous and exergonic reaction). • When a reaction is pushed away from equilibrium, the free energy of system increases (non-spontaneous and endergonic reaction). • When a reaction reaches equilibrium, ΔG = 0, because there is no net change in the system.

15. Free energy and metabolism • Reactions can be classified based upon their free energy changes: • Exergonic reaction – a reaction that proceeds with a net loss of free energy. • Endergonic reaction – an energy-requiring reaction that proceeds with a net gain of free energy; a reaction absorbs free energy from its surroundings.

16. Free energy and metabolism Exergonic reaction Chemical products have less free energy than the reactant molecules. Reaction is energetically downhill. Spontaneous reaction. ΔG is negative. -ΔG is the maximum amount of work the reaction can perform Endergonic reaction Products store more free energy than reactants Reaction is energetically uphill. Non-spontaneous reaction (requires energy input) ΔG is positive. +ΔG is the minimum amount of work required to drive the reaction.

17. Free energy and metabolism • If a chemical process is exergonic, the reverse process must be endergonic. • For example: • For each mole of glucose oxidized in the exergonic process of cellular respiration, 2870 kJ are released (ΔG = -2870 kJ/mol or -686 kcal/mol). • To produce a mole of glucose, the endergonic process of photosynthesis requires an energy input of 2870 kJ (ΔG = +2870 kJ/mol or +686 kcal/mol).

18. How a free-energy gradient keeps metabolism away from equilibrium: a hydraulic analogy.

19. the structure of ATP and • (b) the hydrolysis of ATP to yield ADP and inorganic phosphate. ATP

20. The Nature of ATP • ATP is the immediate source of energy that power cellular work. • ATP powers three main kinds of work in a cell: • mechanical work, cilia beating, the contraction of the muscle cells, the movement of the chromosomes during cellular reproduction; • transport work, the pumping of substances across membranes against the direction of spontaneous movement; • chemical work, the pushing of endergonic (absorbing free energy from the surrounding) reactions that would not occur spontaneously, such as the synthesis of polymers from monomers.

21. Energy coupling by phosphate transfer. In this example, ATP hydrolysis is used to drive an endergonic reaction, the conversion of the amino acid glutamic acid (Glu) to another amino acid, glutamine (Glu--NH2). (a) Without the help of ATP, the conversion is non-spontaneous.

22. Energy coupling by phosphate transfer. (b) As it actually occurs in the cell, the synthesis of glutamine is a two-step reaction driven by ATP. The formation of a phosphorylated intermediate couples the two steps. 1. ATP phosphorylates glutamic acid, making the amino acid less stable. 2. Ammonia displaces the phosphate group, forming glutamine.

23. Energy coupling by phosphate transfer. (c) Adding the delta G for the amino acid conversion to the delta G for the ATP hydrolysis gives the free-energy change for the overall reaction. Because the overall process is exergonic (has a negative delta G), it occurs spontaneously.

24. ATP formation We can write the chemical reaction for the formation of ATP as: a) in chemicalese: ADP + Pi + energy ----> ATP b) in English: Adenosine diphosphate + inorganic Phosphate + energy produces Adenosine Triphosphate The chemical formula for the expenditure/release of ATP energy can be written as: a) in chemicalese: ATP ----> ADP + energy + Pi b) in English Adenosine Triphosphate produces Adenosine diphosphate + energy + inorganic Phosphate

25. How to Make ATP • Two processes convert ADP into ATP: • 1) substrate-level phosphorylation; and • 2) chemiosmosis. • Substrate-level phosphorylation occurs in the cytoplasm when an enzyme attaches a third phosphate to the ADP (both ADP and the phosphates are the substrates on which the enzyme acts). Enzymes and the formation of NADH and ATP.

26. ATP regeneration ATP is renewable and can be regenerated by the addition of phosphate to ADP Exorgenic reaction – catabolic reaction providing energy for ATP making ATP Energy from catabolism Energy for cellular work i ADP + P Endorgenic reaction Requires G = +7.3 kcal/mol

27. Enzymes Catalyst – chemical agent that accelerate a reaction without being permanently changed in the process. Enzymes are catalytic proteins that change the rate of a reaction without being consumed by the reaction. The initial investment of energy for starting a reaction – the energy required to break bonds in the reactant molecules – is known as the free energy of activation, or activation energy. Transition state – unstable condition of reactant molecules that have absorbed sufficient free energy to react.

28. An energy profile of a reaction The reactants AB and CD must absorb enough energy from the surroundings to surmount the hill of activation energy (EA) and reach the unstable transition state. The bonds can then break, and as the reaction proceeds, energy is released to the surroundings during the formation of new bonds.

29. An energy profile of a reaction This particular graph profiles the energy inputs and outputs of an exergonic reaction, which has a negative ΔG; the products have less free energy than the reactants.

30. Enzymes lower the barrier of activation energy Without affecting the free-energy change (delta G) for the reaction, an enzyme speeds the reaction by reducing the uphill climb to the transition state.

31. Enzymes are substrate-specific The reactant an enzyme acts on is referred to as the enzyme’s substrate. Substrate(s) Product(s) Enzyme Glucose H2O Sucrose Sucrase + Fructose

32. Catalytic cycle Substrate (sucrose) Active site Enzyme (sucrase) Fructose Enzyme and substrate are available Glucose 1 Products are released 4 Enzyme-substrate complex H2O Substrate is converted to products Substrate binds to enzyme 3 2

33. Environmental factors affecting enzymes Optimal temperature for typical human enzyme Optimal temperature for enzyme of thermophilic (heat-tolerant) bacteria 20 40 60 80 100 Temperature (C) Rate of reaction Optimal pH for trypsin Optimal pH for pepsin 0 1 2 3 4 5 6 7 8 9 10 pH

34. Cofactors Cofactors are nonprotein helpers for catalytic activity; they are either bound tightly to the active site or bound loosely and reversibly along with the substrate. Cofactors may be either inorganic (Zn, Fe, Cu) or organic (specifically called a coenzyme). Most vitamins are coenzymes.

35. Substrate Active site Inhibitors (a) A substrate can normally bind to the active site of an enzyme (b) A competitive inhibitor mimics the substrate and competes for the active site Competitive inhibitor (c) A noncompetitive inhibitor binds to the enzyme at a location away from the active site, but alters the conformation of the enzyme so that the active site is no longer fully functional Noncompetitive inhibitor

36. The control of metabolism The molecules that naturally regulate enzyme activity in a cell act like reversible noncompetitive inhibitors by binding to an allosteric site. Allosteric site Active site Inactive form Active form (a) Conformational changes in an allosteric enzyme

37. The control of metabolism Inhibitor Activator Active form stabilized by an allosteric activator molecule Inactive form stabilized by an allosteric inhibitor molecule (b) Allosteric regulation

38. Initial substrate (threonine) Feedback Inhibition Enzyme 1 (threonine deaminase) Intermediate A Enzyme 2 Intermediate B Enzyme 3 Feedback inhibition loop (end-product is allosteric inhibitor of enzyme 1) Intermediate C Enzyme 4 Intermediate D Enzyme 5 End-product (isoleucine)

39. Cooperativity In an enzyme molecule with multiple subunits, the binding of one substrate molecule to the active site of one subunit causes all the subunits to assume their active conformation, via the mechanism of induced fit. Substrate Active form stabilized by an allosteric activator molecule Active form stabilized by a substrate molecule

40. Enzymes localization Each organelle contains certain number of enzymes that carry out specific functions. Some enzymes and enzyme complexes have fixed locations within the cell as structural components of particular membrane. Others are in solution within specific membrane-enclosed eukaryotic organelles. For example, in eukaryotic cells the enzymes for cellular respiration reside within mitochondria.

41. Conclusions • ATP powers cellular work by coupling exergonic reactions to endergonic reactions: it drives endergonic reactions by transfer of the phosphate group to specific reactants – cells can carry out work; cell respiration (catabolic pathway) drives the regeneration of ATP from ADP. • Enzymes speed up metabolic reactions by lowering energy barriers (activation energy EA). • Enzymes are substrate-specific. • The active site is an enzyme’s catalytic center • A cell’s physical and chemical environment affects enzyme activity. • Metabolic control often depends on allosteric regulation • The localization of enzymes within a cell helps order metabolism

42. Reading Ch. 8 (142-161)