Principles of Metabolism

1. Principles of Metabolism

2. Some fundamental concepts • Energy • Potential vs kinetic • Chemical vs electrical • Work • Moving material from place to place • Creating order and structure • Power – rate of doing work

3. Here comes the Law • First Law – mass and energy are conserved • Second Law – seen as a whole, a system always proceeds in the direction of more disorder (entropy) and less ability to do work (Gibbs free energy)

4. Chemical equilibrium • Specifically, systems approach thermodynamic equilibrium by giving up some of their potential to do work and increasing their disorder. These facts determine the direction in which a chemical reaction will proceed spontaneously, and the ratio of reactants and products at equilibrium.

5. Cells are not systems at equilibrium • In order to drive reactions that create and maintain order and structure, cells must couple these processes to energy-yielding reactions. As a whole, a cell is far from equilibrium and is operating as an open system in steady state.

6. Role of enzymes in cellular processes • Increase the rate of spontaneous reactions by lowering their activation energy. • Couple energy yielding (exothermic) reactions to energy requiring (endothermic) reactions.

7. Lowering the activation energy of a spontaneous reaction

8. Coupling of reactions: a mechanical analogy coupled uncoupled

9. There are a number of important mediators of energy coupling • Carriers of phosphorylation potential (ATP, creatine phosphate, GTP, UTP, etc.) • Carriers of redox potential (NADH and FADH2 are participants in catabolic processes, NADPH is involved in anabolic processes like fatty acid synthesis) • Gradients of electrical charge, which can apply electrical driving force to ions (molecules or atoms that possess an electrical charge) • Gradients of chemical concentration, in which dissipation of the gradient releases energy that is used to drive an energy-requiring process • Electrochemical gradients, in which the total energy available is the sum of the electrical driving force and the chemical driving force.

10. the different energy couplers are interconvertible • For example, as we will see in oxidative energy metabolism, most of the potential energy of foodstuff is captured first as reduced coenzyme (NADH, FADH2), then converted to an electrochemical gradient of H+ across the mitochondrial inner membrane, and then converted to ATP. • Or in another example, the Na+ /K+ pump in the plasma membrane is powered by ATP and creates an electrochemical gradient of Na+ directed toward the cell interior - diffusive leakage of Na+ back into the cell can be coupled to energize uptake of glucose (some cell types) or amino acids (almost all cell types).

11. Of all the couplers, ATP is the nearest thing to a universal energy currency in cells • In ATP and ADP, a lot of free energy is stored in the electrostatic repulsion of the adjacent O- , so these are sometimes called high-energy phosphate bonds.

12. ATP hydrolysis in cells • In the cell, the ΔG of the ATP hydrolysis can be even larger than the standard ΔG° of -7.3 kcal/mol • ΔG = ΔG° + RT ln ([ADP][Pi]/[ATP]) • Standard conditions: [reactants] = 1 mol/l: ΔG = ΔG° • In cells the [ATP] is normally much higher than [ADP]; in muscle [ATP] = 4 mM; [ADP]=0.013 mM • ΔG = ΔG° + RT ln ([ADP][Pi]/[ATP]) = -12 kcal/mol • Note: under these conditions in cells it also takes more energy to rephosphorylate ADP! If you got more energy out, you had to also be putting more in. • If the system were in equilibrium (ΔG = 0), [ADP] would be over 105 times greater than [ATP] • ΔG0 = -RT ln K • K= 10 –ΔG°/ 2.303 R T • K= 10-ΔG°´/ 5.70 • K=2.2 x 105 K is the equilibrium constant

13. ATP and its relatives • Apart of other nucleoside phosphates that are used in metabolism as energy carriers, the structural motif of ATP returns in other important molecules: • NAD+/NADH + H+ and FAD/FADH2 serve as messengers/carriers for reduction equialents (e- + H+) • A + e- + H+----> AH, • is a hydrogenation and is a reduction. The opposite is a dehydrogenation reaction and is an oxidation.

14. ATP and its relatives • Apart from other nucleoside phosphates that are used in metabolism as energy carriers, the structural motif of ATP returns in other important molecules: • Coenzyme A serves as a messenger/carrier of acetyl groups

15. The cellular store of ATP is small and turns over rapidly • A 70 kg person might consume 2800 kcal/day (=11,700kJ/day) of foodstuff • If total energy metabolism operated at 50% efficiency and none of the calories were stored as fat or glycogen, and • 1 mole of ATP yielded 50kJ of free energy • About 117 moles or about 64 Kg of ATP would be hydrolyzed per day, for a turnover rate of 1300/molecule/day • Exertion would drive this value up substantially – a 2 hour run could cost 60 moles of ATP.

16. To ensure that ATP can turn over rapidly, it should not have to travel far – ideally, only a few microns at most! This is an example showing the close placement of a mitochondrion to the contractile machinery in a skeletal muscle cell

17. This is another example, in which mitochondria actually move to the site of the energy demand. On the left is a cell of non-stimulated Malpighian tubule of the blood-feeding bug Rhodnius. On the right is a portion of a cell from a stimulated tubule. Mitochondria are entering the microvilli to be as close as possible to the apical membrane, the site of an energy demanding ion transport process that is turned on when the bug is eliminating the fluid and ions taken on in a blood meal.

18. Information and energy are interconvertible • Maxwell’s Demon – a simple example • ATP and other phosphorylated molecules are frequently information carriers as well as energy carriers – for example, there is a large category of G-proteins (GTP-binding proteins) that serve as intracellular signals, and phosphorylating a protein is a universal mechanism of controlling that protein’s cellular function.

19. Metabolism consists of catabolism and anabolism • Foodstuffs and cellular energy reserves consist of complex molecules rich in reducing power – the ability to give up electrons and become oxidized. • Catabolism (by mostly exergonic reactions) leads to the creation of disorder in the cell - production of simpler, energy-poor endproducts: carbon dioxide, water, ammonia – sometimes with capture of free energy either directly as ATP or as reduced coenzyme (NADH, FADH, NADPH) • Anabolism is biosynthesis – it involves creating additional order in the cell - new covalent bonds are formed – energy is provided by ATP and NADPH.

20. 4 ways of stating the same energetic truth • Irreversible reactions make metabolic pathways directional – water that has fallen over a waterfall cannot turn around and go back • Every metabolic pathway has a first committed step – early in the pathway, an exergonic reaction step commits its product to continue in the pathway • The same pathway usually cannot operate in either the catabolic or anabolic direction – instead, there must be a detour around each exergonic reaction step in the pathway. • Every pathway has a rate-limiting step – the choke-point step in a reaction sequence is one at which reactants and products are far from equilibrium with each other and the enzyme is almost constantly saturated.

21. Control of metabolic pathways • Allosteric control – substrates and/or products of the pathway feed back on a rate-limiting enzyme • Covalent modification – the rate constants of individual enzyme molecules may be changed by phosphorylation, binding/unbinding of a control protein, bonding of Ca++, or subunit assembly/disassembly. • Movement of enzymes between an active to an inactive pool. • Genetic control – induced synthesis of more or less enzyme, or of an alternative enzyme.

22. A little review of redox chemistry

23. Oxidation/reduction • Oxidation is not only the addition of oxygen atoms • Oxidation refers to the removal of electrons • Reduction is the addition of electrons • Example: Fe2+ is oxidized if it loses an electron to become Fe3+.

24. Oxidation/reduction • This term also applies to shifts of electrons between atoms linked by a covalent bond.

25. When carbon is covalently bonded to an atom with a strong affinity for e-, such as O, Cl, or S, it gives up more than its equal share of electrons. • It acquires a partial positive charge and is said to be oxidized

26. Oxidation of methane (CH4) • C atoms bonded to H have more than their share of electrons and are thus reduced

27. Oxidation of methane • The C atom of CH4 can be converted to CO2 through the successive removal of its H atoms. • With each step, e- are shifted away from C and C becomes progressively more oxidized

33. Hydrogenation/dehydrogenation • Often when a molecule picks up an e- it also picks up a H+ at the same time. • A + e- + H+----> AH • This is a hydrogenation reaction and is a reduction. • The opposite is a dehydrogenation reaction and is an oxidation.

34. “Life is nothing but an electron looking for a place to rest.” Albert Szent-Gyorgi Szent-Gyorgi received the Nobel Prize for Physiology and Medicine in 1937. He was the first to isolate Vitamin C. His discoveries provided the basis for the discovery of the “citric acid cycle”, the substrate reactions of oxidative metabolism.

35. An Overview of the 3 Stages of Energy Metabolism • 1st stage: Large molecules in food are broken down into smaller units. This is a preparation stage without capture of energy. • Proteins -> amino acids, • Polysaccharides -> monosaccharides (glucose, ...) • Fats -> glycerol, fatty acids.

36. 2nd stage: Molecules are degraded to simple units that play a central role in metabolism. Most of them are converted into the acetyl unit of acetyl CoA. Some ATP is generated in this anaerobic stage, but amount is small compared with 3rd stage.

37. 3rd stage:ATP is produced from the complete oxidation of the acetyl unit of acetyl CoA. Acetyl CoA brings acetyl units into the citric acid cycle, where they are completely oxidized to CO2. Four pairs of electrons are transferred (three pairs to NAD+ and one pair to FAD) for each acetyl group that is oxidized. Then, a proton gradient is generated as electrons flow from the reduced forms of these carriers to O2, and this gradient is used to phosphorylate ADP to ATP.