Introduction to metabolism

1. Introduction to metabolism Biochem I Lecture 2 Sept. 12 / 06 Chap 15

2. “Metabolism” is the dynamic regulation of the cell’s environment • This requires the action of proteins that are biological catalysts (e_______) acting together in pathways • We will go into these in more detail when we consider synthesis and degradation of the four classes of biomolecules • These pathways can be placed in two broad classes: either • build molecules from less complex building blocks through anabolism, an en_____ processes • Pathways that require energy input in order to proceed or • break complex molecules down via catabolism, which is ex______. • Pathways that convert energy into biologically useful forms • Metabolism is the sum total of these enzyme reactions in the cell: a highly integrated network of chemical reactions

3. Cells are alive because they control their energy use • Now is a good time to introduce concepts concerning how cells are able to carry out these reactions, for example • How enzymes do their work and their activity is regulated • How molecules and ions can cross membranes • These concepts really can be reduced to how the cell manages energy: balancing the energy obtained from catabolism with the energy required for the synthesis of new molecules in anabolism • How cells extract reducing power and energy from their environment • How building blocks of molecules can be synthesized and then assembled into more complex molecules that possess specific roles in the cell

4. Ingested carbohydrates and lipids provide energy for metabolism From Agriculture and Agri-Food Canada website • The total number of reactions (and enzymes) required for metabolism is 1000+ even in “simple” organisms like E. coli • Taken as a whole, metabolism seems random and chaotic • However, the way fuel molecules are metabolized to create the “molecules of life” follows a common pattern in all living organisms Fig 15.2

5. Biochemical unity extends to metabolism • Metabolism has a coherent design containing many common motifs, for example • A common energy currency • Limited number of activated intermediates that reappear in many pathways • These are shown by branchpoints between distinct pathways in diagrams of metabolism • ~100 common molecules play a central role • Complex metabolic web but consists of • Common types of reactions catalyzed • Simple reaction mechanisms • Common pathway regulation mechanisms This commonality considerably simplifies the study of metabolic pathways!

6. Continual input of free energy is required by living organisms • This free energy powers • Cellular movement (muscle contraction, intracellular movement of organelles and cytosol) • Active transport of molecules and ions across plasma and organelle membranes • FAs, proteins for glycosylation and cellular targeting • Biomolecule synthesis • Remember The First Law of Thermodynamics: energy can be neither ______ nor ______, just ______. • Two types of organism that can change available energy to forms useful for cellular work ( G_____ free energy) • Phototrophs (autotrophs) that use _____ energy to transform simple molecules to biomolecules • The cardinal source of energy on the planet: remember where humans get half of their dietary CHO! • Chemotrophs obtain their energy through oxidation of food • Oxidation of carbon compounds results in chemical energy (ATP, reducing power)

7. Metabolism allows cells to shift between forms of energy • This energy can then be transformed into other types to do cellular work • Mechanical energy • to move molecules from one place to the other (e.g., across membranes) • to synthesize biomolecules • Electrical energy to convey information through nerve impulses • Converted to further forms of chemical energy! • Metabolism is a linked series of reactions transforming one compound (substrate) into another (product) • One example is glucose metabolism Fig 15.1

8. Pathways must satisfy two requirements • One: Individual reactions must be specific • An enzyme must make one product required by the next step in the pathway • Two: The set of reactions in the pathway must be thermodynamically favored • That is, the change in free energy for the pathway, ΔG, must be ______. • Only under this condition can the reaction occur spontaneously • For a reaction A + B ↔ C + D, the ΔG of this reaction is given by Standard free energy change at STP Gas constant: 2 x 103 kcal/(mol . degree) In Kelvin

9. Coupling allows thermodynamically unfavorable reactions to occur in vivo • Note that the free energy change in a reaction is dependent on the equilibrium constant K (a/k/a K’eq) since K’eq = conc of products / conc of substrates = [C][D] / [A][B] • Thus the free energy liberated in a chemical reaction depends on • The nature of the substrates and products (included in the ΔGo term), and • Their concentrations (second term) • Thermodynamically unfavorable reactions can be made to occur by coupling reactions with a positive ΔG to driving reactions that possess a negative ΔG Energetically unfavored endergonic rxn… …can be coupled to a spontaneous exergonic rxn… This can be used to drive the entire rxn forward!

10. ATP hydrolysis is used as the exergonic driver in endergonic pathway steps • ATP • a highly accessible common energy currency • The free energy donor facilitating energetically unfavored, endergonic biosynthetic pathways • An adenosine phosphate or adenylate: an adenine base, a cyclized ribose sugar (furanose), and a triphosphate unit • ATP can be hydrolyzed to ADP or AMP by cleaving one or the other phoshphoanhydride bonds Fig 15.3 AMP ADP High energy phosphanhydride bonds

11. ATP’s ubiquity means that it is the cell’s energy currency • Actual free energy change close to -50 kJ / mol in vivo • ATPADP conversion most typical; the fundamental mode of energy transfer (transduction) in biological systems • Other nucleotide phosphates are involved in energy transduction • Energetically equivalent and interchangeable but ATP is favored form • Electron carriers NAD+ and FAD are derivatives of ATP Thus the role of ATP in energy production is of cardinal importance NAD structure from http://www.agen.ufl.edu/~chyn/age2062/lect/lect_02/of2_13b.gif

12. ATP hydrolysis shifts the equilibrium of reactions to which it is coupled • Biosynthetic reactions are generally energetically unfavorable • The equilibrium constant for a coupled reaction is the product of the K’eq of the individual reactions • For the endergonic reaction A↔B, suppose ΔG°’ (ΔG at STP) = +16 kJ / mol • Given this information, K’eq can be calculated since K’eq = conc of products / conc of substrates = [B] eq / [A] eq • K’eq can thus be calculated from the definition of ΔG = ΔG°’ + RT ln ( [product] / [substrate] ) • Can walk thru the derivation on p. 209 of the text if desired, but simply, K’eq = 10^(- ΔG°’ / 1.36) • Thus, given the ΔG°’ for any reaction, we can calculate K’eq • For ΔG°’ = +16 kJ / mol, K’eq = 1.2 x 10-3

13. Coupling ATP hydrolysis to an endergonic reaction drives it toward product formation • If the hydrolysis of ATP is added to the reaction, the free energy change of this new coupled reaction is the sum of the individual reactions: 16 + (-30.5) ~ 14 kJ / mol and the K’eq value for this coupled reaction is The ratio of [B] / [A] is affected by the coupling of the reaction; rearrange to get So coupling changes the P/S ratio of the rxn: Coupling this reaction to ATP hydrolysis serves to change a reaction essentially at equilibrium to one significantly favoring product formation (an increase of 108 fold, from 1.2 x 10-3)! ATP levels are kept high by the cell: 500 M-1

14. ATP possesses a high phosphoryl transfer potential • In a series of reactions coupled to ATP hydrolysis, the equilibrium ratio changes by 108n, where n is the number of ATP molecules hydrolyzed in the pathway • Thus when a series of reactions hydrolyzes 3 ATP, the equilibrium ratio favors product formation 1024 fold more than in the individual uncoupled reactions • In the equation A ↔ B, A and B do not necessarily represent different chemical species • They may be • Ion concentrations inside and outside a membrane • Different conformations of the same protein or compound (plus or minus an activating phosphate group, for example in the molecular motor proteins such as actin and myosin that drive muscular contractions) Terminal gamma phosphate Fig 15.3

15. ATP can readily donate its terminal gamma phosphate to other molecules • This is a common way to regulate metabolism! • ATP is a high energy compound because it has an extremely high phosphoryl transfer potential • What makes ATP so reactive? • The number of resonance forms of the terminal γ-phosphate of the ATP structure of the triphosphate group is limited • 3 Pi groups contain a highly negative charge in close proximity: repel each other • ATP cannot bind water as effectively as ADP • All favor ADP versus ATP in the environment of the cell • Many other compounds also readily give up their phosphate groups and have a high phosphoryl transfer potential Table 15.1 Most of these phosphorylated compounds are important in linking primary carbon metabolism to energy production !

16. ATP is an immediate energy source for metabolism • Long term energy sequestration is via lipid storage • 100 g of ATP in the body, turnover is very high • May use 500 g / min during strenuous exercise = 60 kg during a 2-h run • Thus generation of ATP is the primary role of metabolism • Remember that oxidative metabolism reduces sugar to CO2, and the last e- acceptor in oxidative phosphorylation (the primary means of synthesizing ATP) is O2, which is reduced to H2O Fig 15.8 6 glucose (C6H12O6) + 6 O2  6 CO2 + 6H2O

17. Energy extraction means oxidation • The more reduced a substrate, the more energy yield from it since each oxidation step releases energy and can be coupled to ATP synthesis Most reduced Most oxidized Fig 15.9 We say that highly reduced molecules contain a lot of potential energy, but how is that energy extracted and used to fuel ATP synthesis?

18. Extracting energy from the diet There are three stages in the extraction of ATP from food, first described by Hans Krebs, a pioneer in carbohydrate metabolism (the TCA cycle is a/k/a the Krebs cycle!) • Stage I reduces food to molecule precursors: lipids to FAs and glycerol, protein to amino acids, polysaccharides to monosaccharides Fig 15.12 • Stage II oxidizes these building blocks to small metabolites present in many metabolic pathways: FAs, glucose, amino acids to acetyl unit of acetyl CoA • Stage III oxidizes acetyl CoA to CO2 in common pathways in converting the potential energy of fuel molecules into useful energy

19. Stage III of energy extraction sequentially oxidizes common metabolites derived from food to make ATP • This last stage takes advantage of the electrochemical potential of gradients created by different concentrations of protons on either side of a lipid membrane • a/k/a oxidative phosphorylation across the inner membrane of mitochondria • Oxidation of carbon fuel releases hydrogen = protons • Where does this take place? In the mitochondrial matrix (inside the inner membrane) • This creates a proton gradient relative to the cytosol, H+ leave • This reverses the proton gradient and allows ATP synthesis This process catalyzes 90% of ATP synthesis! Oxidation of fuels pumps H+ out Inner membrane Fig 15.11 Mitochondrial matrix Influx of H+ forms ATP Fig. 15.11

20. Unifying metabolic themes are common in all organisms and support the concept of biochemical unity • Common metabolites, reactions and regulatory schemes indicate a common evolutionary heritage • Let’s list some of these common themes 1. Common activated e- carriers power metabolism (a) Fuel oxidation • ATP is an activated carrier of phosphoryl groups • NAD+ and FAD are activated electron carriers (b) Reductive biosynthesis • Need reducing power because the substrates are more oxidized than the products: acetyl CoA versus FAs • Usually as NADPH, not NADH: the phosphate is a ‘tag’ that enables enzymes to leave NADH as an e- carrier for oxidative phosphorylation instead of biosynthetic reactions

21. Common electron carriers also include metabolites with two carbon atoms (c) Two carbon fragments • Coenzyme A carries acetyl groups for biosynthesis and catabolism (feeds into the TCA cycle) • Carries a high ΔG°’ for hydrolysis comparable to that of ATP (-30.5 kJ / mol); transfer of the acetyl group is highly favorable thermodynamically • Notes: • All activated carriers are not easily oxidized (NADH, FADH2, NADPH) or hydrolyzed (ATP, acetyl CoA) in the absence of an enzymatic catalyst • This kinetic stability allows enzymes to control the flow of free energy and reducing power • Same e- carriers in all organisms! Metabolism is modular!

22. Unifying theme 2: A limited number of reaction types make up metabolism • Thousands of enzyme reactions can be reduced to just six types (summarized in table 15.5) (a) Redox • Passing e- between molecules • Can also be used to generate useful energy as reducing power (b) Ligation • Form covalent bonds, assisted by ATP hydrolysis; common in anabolism

23. The common six reaction mechanisms in biochemistry (c) Isomerization • Rearranges atoms within a molecule; reorients double bonds (d) Group transfer e.g., in ATP synthesis from ADP! • Or in the phosphorylation of monosaccharides to activate them

24. The six common reaction mechanisms in biochemistry (e) Hydrolytic • Cleave covalent bonds by adding water • Used to break down large molecules • e.g., to break down peptides composed of multiple amino acids to smaller peptides in protein catalysis (f) Addition/removal of functional groups to/from double bonds • The enzymes performing these reactions are classified as lyases • Include dehydrations to form double bonds • Reaction rules: • Can proceed in either direction • Same sequence of reactions occurs in multiple pathways • Compare FA degradation to TCA cycle: oxidation, hydration, oxidation! • Metabolism possesses an underlying chemical simplicity (and elegance!)

25. Metabolic control is the study of the regulation of enzyme activity Extensive regulation of the activity of enzymes in the metabolic network is a must! • Must match demand for the product of a pathway with the availability of its substrate(s) • This control must be flexible to accommodate microsecond-to-microsecond changes in demands for energy The reaction rate of enzymes can be controlled through a variety of mechanisms…

26. 2 major mechanisms to control reaction rate • Coarse control of enzyme amount (concentration) • Regulated by changing the transcription rate of the genes coding for the enzyme • Gene on DNA  gene specific mRNA  protein • Gene transcription can be quickly upregulated (or protein can be broken down into its constitutive amino acids), but this is the slowest (most energetically demanding and least elegant!) way of changing enzyme activity • Fine control: controlling enzyme activity • Principle: Keep the enzyme amount the same, but change its reaction rate (how many molecules of substrate it can convert to product in a given time) • Allosteric control / feedback inhibition: the product of a pathway inhibits the enzyme(s) that make it • Change pH: this affects enzyme activity • Reversible covalent modification: add functional groups to proteins to change their activity (e.g., Pi) • Compartmentation of substrates in the cell: sequester substrates in an organelle to shut down a cytosolic pathway • … and other mechanisms we will explore further when we examine enzymes in more detail

27. Energy charge regulates cellular metabolism • Metabolism is absolutely dependent on the high availability of ATP in the cell • A measure of the relative amount of nucleotide phosphate in the cell is useful to determine how “charged” the cell is for performing useful work • It is a measure of the number of high energy phosphoanhydride bonds available to be hydrolyzed versus the total cellular amount of ATP, ADP and AMP • The energy charge can range from 0 (all AMP = d____!) to 1 (all ATP) • Healthy cells typically measure between 0.80 and 0.95

28. Cellular metabolism constantly adjusted to favor high ATP concentration while not wasting G • Logically, catabolic pathways that generate ATP are inhibited at a high energy charge, while anabolic pathways that require coupling energy to proceed are inhibited at low energy charge • Curves of activity versus energy charge intersect at about 0.9: high energy charge is the “normal” state of a cell! • Just like pH, the cellular energy charge is buffered! • Favors a high value! Fig 15.19