1. Fundamentals of Biochemistry Third Edition Fundamentals of Biochemistry Third Edition Chapter 14 Introduction to Metabolism Chapter 14 Introduction to Metabolism Copyright © 2008 by John Wiley & Sons, Inc. Donald Voet Judith G. Voet Charlotte W. Pratt

2. These are all the biochemical pathways: do you recall the Prime Radiant in Isaac Asimov’s Second Foundation? Well, it’s kind of like that. Metabolism is a combination of anabolism and catabolism

3. Different portions of metabolism occur at different places in the cell

4. Vitamins are organic micronutrients that are not synthesized by animals, and therefore must be part of the diet. Minerals and trace elements are inorganic micronutrients.

6. Metabolites are any of the reactants, enzymes or products in any metabolic pathway. There are about 4000 such reactions. Broadly, degradation reactions generate free energy, stored in the bonds of an energy- storing molecule, like ATP. Biosynthesis reactions, by contrast, require free energy release by the use of energy-storing molecules.

7. Clearly, there should be systematic way of understanding metabolism. In fact, we will examine three ways: 1.The materials of metabolism: reactants, intermediates and products 2.The free energy changes of metabolism 3.The control of metabolism, which is to say enzymatic control

8. The materials: shown by the blue boxes, starting with the three major classes of biochemicals, and showing their fates

9. The free-energy changes: energy-storing molecules (called energy carriers) are shown by the ovals and explosions

10. The control: A portion of the light and dark mechanisms in a plant shown with the controlling enzymes (green boxes)

11. All of these ways of understanding metabolism are connected by thermodynamics Given an equilibrium: A + B C + D At equilibrium: Most biochemical reactions are near equilibrium Recall ΔG°’ means “free energy change at physiological standard conditions”

12. Some biochemical reactions operate at far from equilibrium conditions 1.Metabolic pathways are irreversible 2.Every metabolic pathway has a committed first step 3.Catabolic and anabolic pathways are different

13. Metabolic flux is the amount of metabolite that undergoes a process over a given time: controlling the activity of enzymes regulates the flux. Allosteric control: For instance, feedback inhibition, in which the product of a process binds to one of the early steps’ enzymes and alters its active site.

14. Metabolic flux is the amount of metabolite that undergoes a process over a given time: controlling the activity of enzymes regulates the flux. Allosteric control: For instance, feedback inhibition, in which the product of a process binds to one of the early steps’ enzymes and alters its active site. Covalent modification: For instance, the phosphorylation or dephosphorylation by kinases and phosphorylases that allow enzymes to be more or less active.

15. Substrate cycling: Having different enzymes for the forward and backward reactions allows for fine-tuning of the overall flux.

16. Genetic control: The most gross control of all is the number of enzyme molecules present, which is controlled by how quickly and often a gene is expressed

17. Energy carriers have high-energy bonds: that is, bonds, when broken, release a relatively large quantity of free energy. The phospho-anhydride bonds in ATP are a good example.

18. base moiety sugar moiety

19. ATP hydrolysis is represented symbolically in many ways; for instance: Or as the textbook does: ATP + H 2 O ADP + P i ATP + H 2 O AMP + PP i where P i = inorganic phosphate = PO 4 3– and PP i = pyrophosphate = P 2 O 7 4–

20. Phosphate and pyrophosphate are particularly stable due to resonance, which means that any process that releases a free phosphate or pyrophosphate will also release a lot of free energy.

21. The release of free energy of ATP hydrolysis can be coupled to an endergonic reaction to drive that reaction against the energy barrier. We did warn you that Hess’s Law (Chem 162) was useful!

22. The pyrophosphate can be further hydrolyzed to phosphates, and that reaction may also be coupled to drive an endergonic reaction forward. “An average person at rest consumes and regenerates ATP at a rate of ~3 mol (1.5 kg) per hour and as much as an order of magnitude faster during strenuous activity.”

23. In fact, phosphorylating other metabolites can make them high-energy compounds: this is called substrate- level phosphorylation.

24. So why are these considered “low-energy” compounds?

25. Because their products do not have significantly different resonance structures compared to the original compound, so there is little difference in free energy content.

26. Whereas these compounds do!

27. On a related note, nucleotide triphosphophates (NTPs) can freely interconvert: for instance, ATP + NDP ADP + NTP Adenylate kinase is shown (note the extreme conformational change between T and R states.

28. In addition to phosphoester compounds, thioester compounds may also be high- energy. The text uses the convention that a bond shown as “ ~ “ is a hydrolyzable high-energy bond.

29. Ultimately, though, in addition to the harvest of free energy from glucose, respiration (as well as other metabolic reactions) is an oxidation- reduction (redox) process. Thus, this story will become one of how to get electrons from one molecule to another.

30. Recall LEO GER: Loss of Electrons is Oxidation Gain of Electrons is Reduction

31. Recall LEO GER: Loss of Electrons is Oxidation Gain of Electrons is Reduction Iron is being reducedCopper is being oxidized

32. Carbon may also be oxidized and reduced. In glucose, most of the carbon atoms have an oxidation number of zero. In carbon dioxide, the carbon atom has an oxidation number of +4, meaning that a typical carbon atom in glucose will lose four electrons to become the carbon atom in CO 2. Those four electrons must be picked up by another substance, and that turns out to be O 2, which will be reduced to H 2 O,

33. What physically moves the electrons from one substance to another? Electron carriers like NAD. Its reduced form is the high-energy form because it has gained two electrons. Another way to say this is that oxidation is a spontaneous process.

34. Flavin adenine dinucleotide (FAD) is another electron carrier The difference between NAD and FAD is that FAD can accept one or two electrons at a time (NAD may only accept two). Like NADH, FADH 2 is the highest energy form.

35. Recall that every redox couple can be decoupled into two half-cell equations: one for the oxidation and one for the reduction. Because electrons are used to balance the charge in each half-cell equation, a potential for each half-cell can be measured. The potential is measured in volts (V) and is given the symbol E.

36. The calculation of the potential change ΔE of a biochemical reaction not only depends on a Hess’s Law- like addition of half-cell potentials, but also on the factors like the temperature and concentrations of reductants and oxidants. In summary, given a redox reaction: the Nernst equation (Walther Nernst, 1881) will determine ΔE: where ΔE°’ is the potential change at physiological standard conditions, R is the gas constant 8.314 J/molK, n is the number of electrons transferred and F is Faraday’s constant 96485 J/Vmol

37. Experimental approaches to studying metabolism Tracing metabolites isotopically: here, using 13 C NMR to monitor the conversion of glucose to glycogen by using 13 C at carbon 1 of the glucose

38. Using metabolic inhibitors to determine the order of steps in a process. Here, patients with alcaptonuria cannot further metabolize homogentisate, so it is excreted in urine where it can be identified.

39. Systems biology has changed the manner in which metabolism is studied. Techniques like PCR sequencing have increased the amount of data that can be collected; increases in computational capacity have resulted in “whole cell” inventories of DNA, RNA, proteins and metabolites.

40. “Lab on a chip” analysis of gene expression in yeast