Molecular Biology: Metabolism

Principles of Bioenergentics

  • Bioenergetics/thermodynamics (more in Chemistry section)
    • Free energy/Keq
      • Free energy = ΔG
      • Free energy at standard conditions = ΔG°
      • Negative ΔG = reaction will proceed forward
      • Positive ΔG = reaction will proceed backward
      • At equilibrium, ΔG = 0, forward and reverse reaction occurs at the same rate, reaction comes to an equilibrium
      • Keq = equilibrium constant
      • Keq = forward reaction rate constant / reverse reaction rate constant
        • Keq > 1 means at equilibrium, there's more products than reactants
        • Keq < 1 means at equilibrium, there's more reactants than products
      • Relationship of the equilibrium constant and ΔG°
        • ΔG° = -RT ln(Keq)
        • Keq > 1 means ΔG° is negative, the greater the Keq, the more negative the ΔG°
        • Keq < 1 means ΔG° is positive, the smaller the Keq, the more positive the ΔG°
    • Concentration
      • Le Chatelier's Principle = disrupting a system in equilibrium will cause the system to readjust to reachieve equilibrium
      • Adding a reactant will push more products to form
      • Removing a product will cause more reactants to form products
    • Endothermic/exothermic reactions
      • H = enthalpy
      • ΔH = change in enthalpy
      • Endothermic = positive ΔH = energy is needed as an input/reactant (Eg. making ATP)
      • Exothermic = negative ΔH = energy is released as a output/product (Eg. using/hydrolyzing ATP)
    • Free energy: G
      • G = free energy
      • ΔG = change in free energy
      • ΔG = ΔH - TΔS
      • ΔG depends on both change in enthalpy (ΔH), change in entropy (ΔS), and temperature
    • Spontaneous reactions and ΔG
      • When ΔG is negative, the reaction occurs spontaneously
      • Spontaneity says nothing about how fast it will occur though
      • If activation energy is really high, the reaction may not occur at standard conditions at all
  • Phosphoryl group transfers and ATP
    • ATP hydrolysis ΔG << 0 means ΔG is negative, reaction is spontaneous
    • ATP hydrolysis also releases energy
    • ATP -> ADP + Pi
    • Pi = inorganic phosphate
    • Kinases are enzymes that hydrolyze ATP to transfer a phosphate group to a protein
  • Biological oxidation-reduction
    • You get energy from food by oxidizing them
    • Every oxidation reaction is coupled to a reduction reaction
    • Half-reactions
      • The whole oxidation-reduction reaction can be separated into half-reactions
      • The oxidation half (pyruvate + CoA -> acetyl-CoA + CO2)
      • The reduction half (NAD+ -> NADH)
    • Soluble electron carriers
      • Water soluble: NADH, FADH2, NADPH
      • Fat soluble: membrane proteins in the electron transport chain (FMN, CoQ, iron-sulfur complexes, cytochromes)
    • Flavoproteins = electron carriers in oxidation-reduction reactions = FAD, FMN

Basic metabolism

  • Metabolism consists of two parts: Catabolism and anabolism.
  • Catabolism is breaking stuff down for energy.
  • Anabolism is using energy to build stuff for storage.
  • Another name for metabolism is cellular respiration.
  • Steps of aerobic metabolism (needs oxygen)
    • Glycolysis
    • Oxidative decarboxylation
    • Krebs cycle
    • Electron transport chain.
  • Steps of anaerobic metabolism (don't need oxygen)
    • Glycolysis
    • Alcohol or lactic acid fermentation
  • Complete oxidation of glucose to carbon dioxide and water
    • C6H12O6 + 6O2 => 6CO2 + 6H2O
      • C6H12O6: this is glucose. You get it from your diet
      • 6O2: this is molecular oxygen that you breathe in
      • 6CO2: this is carbon dioxide produced by the Krebs cycle. Both the carbon and oxygen in this CO2 comes from the metabolite (glucose)
      • 6H2O: this is water produced in the electron transport chain. The oxygen comes completely from the molecular oxygen that you breathe in
      • If we were to follow the carbon in the metabolite (glucose), it will end up in carbon dioxide.
      • If we were to follow the oxygen in the metabolite (glucose), it will end up in carbon dioxide.
      • If we were to follow the oxygen you breathe in, it will end up in water.
      • As for the hydrogens, they'll either be in water, exist as protons in solution, or be transferred to some other entity.
      • As we can see, the total reaction involves complete oxidation of the metabolite (glucose) and complete reduction of molecular oxygen.
      • When electrons pass from the metabolite (glucose) to molecular oxygen, energy is released.
      • The electron transport chain harnesses this energy.
    • ATP produced per glucose: theoretically 38 (2 from glycolysis, 2 from citric acid cycle, 24 from ETC), actually ~30

Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

  • Glycolysis (aerobic) = convert glucose (6 carbons) to 2 molecules of pyruvate (3 carbons).
    • Location: cytosol.
    • 2 net ATP made for every glucose (2 input ATP, 4 output ATP).
    • 2 NADH made for every glucose.
    • Occurs under both aerobic and anaerobic conditions.
    • Glycolysis is inhibited by ATP.
    • Feeder pathways: breakdown of glycogen and starch (in plants) forms glucose units, which feeds into glycolysis
  • Fermentation (anaerobic glycolysis)
    • Partial oxidation of metabolite (glucose) to pyruvate
    • 2 net ATP produced per glucose
    • Pyruvate is then reduced to either alcohol or lactate
    • Alcohol fermentation = pyruvate reduced to ethanol = bacteria
    • Lactic acid fermentation = pyruvate reduced to lactate = humans
    • The purpose of anaerobic fermentation is to regenerate NAD+, which is needed for glycolysis
    • 1 NAD+ regenerated for every pyruvate
    • Oxidation-reduction reaction: reduce pyruvate, oxidize NADH
  • Gluconeogenesis
    • It's essentially the reverse of glycolysis
    • Starting material is pyruvate, end product is glucose
    • A lot of the enzymes are the same/shared, they just work in reverse because of opposite reactant/product concentrations (Le Chatelier's principle)
    • Enzymes that are not shared play important regulatory functions
  • Pentose phosphate pathway
    • A shunt that takes glucose-6-phosphate away from glycolysis, makes some new products, and feeds fructose-6-phosphate back into glycolysis
    • Oxidative phase: makes NADPH (used in fatty acid synthesis)
    • Non-oxidative phase: makes ribose-5-phosphate (DNA/RNA synthesis) and erythrose-4-phosphate (aromatic amino acids)

Principles of Metabolic Regulation

  • Regulation of metabolic pathways
    • Regulation tend to occur at:
      • Rate limiting (slowest) enzymes/steps
      • Irreversible reactions
      • Beginning of pathways
    • Products tend to inhibit the enzyme (negative feedback) and lots of reactants tend to activate the enzyme
    • Maintenance of a dynamic steady state
      • When you're living, you are maintaining a dynamic steady state
      • Example of dynamic steady state: cells pump sodium out, but sodium keeps leaking back in, to maintain a low sodium concentration, the cell needs to keep pumping sodium out
      • When you're dead, you would have reached a static steady state
      • Example of static steady state: you're dead, your cell no longer pumps sodium out, sodium is allowed to leak inside as much as it wants to, until it eventually reaches a new steady state with a high intracellular sodium level
      • Similarly, regulation of metabolic pathways serve to maintain a dynamic steady state (eg: exercise = using up more glucose = upregulates the breakdown of glycogen as well as glycolysis)
    • Regulation of glycolysis and gluconeogenesis
      • Insulin = increase glycolysis, suppress glucagon = decrease blood sugar
      • Glucagon = decrease glycolysis, increase gluconeogenesis = increase blood sugar
      • Epinephrine = increase glycolysis (muscle), increase gluconeogenesis = release glucose into blood and use it in muscle = fight or flight
      • Fructose-2,6-bisphosphate: activates glycolysis, inhibits gluconeogenesis
      • High ATP, low ADP = has enough energy, no need to break down for more, but need to store it = inhibits glycolysis, activates gluconeogenesis
      • Low ATP, high ADP = need energy, please break down glucose = activates glycolysis, inhibits gluconeogenesis
      • Regulation occurs at rate limiting enzymes
        • Glycolysis: hexokinase, phosphofructokinase, pyruvate kinase
        • Gluconeogenesis: fructose-1,6-BP, PEP carboxykinase, pyruvate carboxylase
    • Metabolism of glycogen
      • Glycogen phosphorylase: Glycogen -> glucose-1-phosphate
      • Phosphoglucomutase: glucose-1-phosphate -> glucose-6-phosphate
      • Glucose-6-phosphate can either feed into glycolysis, pentose phosphate pathway, convert to glucose (glucose-6-phosphatase)
    • Regulation of glycogen synthesis and breakdown
      • Hormonal: glycogen breakdown promoted by glucagon and epinephrine
      • Hormone -> cAMP cascade -> allosteric effects
      • Allosteric
        • cAMP = active kinase, inactive phosphorylase = promote glycogen breakdown, inhibit synthesis
        • Low cAMP = active phosphorylase, inactive kinase = promote glycogen synthesis, inhibit breakdown
        • Kinase adds phosphate groups, which activates glycogen phosphorylase (breakdown) and inhibits glycogen synthase (synthesis)
        • Phosphorylase removes phosphate groups, which activates glycogen synthase (synthesis) and inhibits glycogen phosphorylase (breakdown)
    • Analysis of metabolic control
      • Identifies what steps in a pathway serve as regulation/control (usually rate limiting steps), and how much so
      • Does this by altering variables (enzyme, metabolite), then seeing how that effects the rest of the pathway
      • Things are quantified (activity of enzyme, concentration of metabolite) and measured so that a precise mathematical model can be generated

Citric acid cycle

  • Acetyl-CoA production (oxidative decarboxylation)
    • Occurs in mitochondrial matrix
    • Converts pyruvate (3 carbons) to an acetyl group (2 carbons)
    • 1 NADH made for every pyruvate
    • Only occurs in the presence of oxygen
    • Acetyl group attaches to Coenzyme A to make acetyl CoA
  • Location: matrix of mitochondria.
  • Acetyl CoA feeds into the cycle.
  • 3 NADH made per acetyl CoA.
  • 1 FADH2 made per acetyl CoA.
  • 1 ATP (GTP) made per acetyl CoA.
  • Coenzyme A is regenerated (during the first step of the cycle).
  • Krebs cycle, TCA, Tricarboxylic acid cycle, citric acid cycle all mean the same thing.
  • Krebs cycle is Inhibited by ATP and NADH.
  • Reactions/substrates/products made simple
    • Citrate -> Isocitrate -> aKG -> succinyl-CoA -> succinate -> fumarate -> malate -> OAA
    • OAA merges with acetyl-CoA to regenerate citrate
    • Enzyme names are intuitive: reactant+dehydrogenase or product+synthase (except succinyl-CoA synthetase - it's the opposite!)
    • Step that generates GTP: succinyl-CoA -> succinate
    • Step that generates FADH2: succinate -> fumarate
    • Steps that generate NADH: isocitrate -> aKG -> succinyl-CoA, malate -> OAA
    • Other metabolic pathways can feed into the citric acid cycle: fat (feeds acetyl-CoA), protein (feeds a lot of places depending on the amino acid)

Oxidative phosphorylation

  • Electron transport chain and oxidative phosphorylation, substrates and products, general features of the pathway
    • Location: the cristae (inner membrane of mitochondria).
    • Substrate: NADH, FADH2
    • Mechanism: Proton gradient
    • The electron transport chain (ETC) is essentially a series of redox reactions (electron transfer), where NADH gets oxidized to NAD+ and O2 gets reduced to H2O
    • The energy released from these reactions generates a proton gradient, which drives ATP synthase to make ATP. This is called oxidative phosphorylation.
  • Electron transfer in mitochondria
    • The series of redox reactions consists of electrons passing from NADH to FMN, to Coenzyme Q, iron-sulfur complexes, and cytochromes (cytochrome b, c and aa3) before finally being used to reduce oxygen
    • NADH is highest in energy, while O2 is lowest in energy. When electrons are passed from NADH down a series of proteins and finally to O2, energy is released.
    • FADH2 is lower in energy than NADH, that's why it releases less energy when it gets oxidized.
    • FADH2 skips FMN and passes its electrons to Coenzyme Q.
  • ATP synthase, chemiosmotic coupling (proton gradient)
    • The energy released from passing electrons down the ETC is used to pump protons into the intermembrane space of the mitochondria.
    • H+ concentration is very high in the intermembrane space (higher than those in the matrix). Thus, this establishes an electrochemical gradient called the proton gradient.
    • H+ wants to migrate down the proton gradient (from the intermembrane space back into the matrix), but it can only do this by going through the ATP synthase.
    • Like a water mill, ATP synthase harnesses the energy of the falling protons (proton motive force) to convert ADP into ATP.
  • Net (maximum) molecular and energetic results of respiration processes: theoretically 38 ATP (2 from glycolysis, 2 from citric acid cycle, 24 from ETC), in reality ~30 ATP
  • Regulation of oxidative phosphorylation: activated by need for ATP (low ATP high ADP)
  • The ETC is inhibited by certain antibiotics, by cyanide, azide, and carbon monoxide
  • Oxidative stress -> mitochondria releasing caspase activators -> caspase cascade -> apoptosis

Hormonal Regulation and Integration of Metabolism (BC)

  • Higher level integration of hormone structure and function
    • Peptide hormones = water soluble = can't pass through cell membrane = bind cell membrane receptors = relays downstream small molecule and kinase cascades = fast (eg. Epinephrine - you need fight or flight to be fast)
    • Steroid hormones = hydrophobic = can pass through cell membrane = goes to the nucleus and regulate gene expression = slow (need time to transcribe mRNA and make proteins, example: sex hormones during puberty)
  • Tissue specific metabolism
    • Brain can only utilyze glucose as energy source
    • Fast twitch (white) muscle fibers primarily use anaerobic respiration (glycolysis)
    • Slow twitch (red) muscle fibers primarily use aerobic respiration (oxidative phosphorylation)
    • The differences in metabolism in different tissues is due to cell differentiation (epigenetic activation/inactivation of genes)
  • Hormonal regulation of fuel metabolism
    • Human growth hormone: increases breakdown of fat and synthesis of protein
    • Cortisol = stress hormone = increases gluconeogenesis and blood sugar
    • Insulin = increases cell uptake of glucose and glycolysis = decreases blood sugar
    • Glucagon = increases gluconeogenesis and breakdown glycogen = raises blood sugar
  • Obesity and regulation of body mass
    • Obesity = dysfunctional regulation of body fat
    • Either a dysfunction where the body refuses to utilize stored fat as an energy source, or a dysfunction in satiety (feeling full after a meal)
    • Obesity causes insulin resistance (metabolic syndrome), which can eventually lead to diabetes

Metabolism of fats and proteins

  • Fat metabolism
    • Location: beta-oxidation occurs in the matrix of the mitochondria. Ester hydrolysis occurs in the cytosol.
    • Fatty esters gets hydrolyzed into free fatty acids by lipases.
    • For example, triacylglycerol gets hydrolyzed into free fatty acids and glycerol.
    • With the help of ATP, the fatty acid is "activated" at the acid end by CoA (to be precise, it turns into a thioester).
    • A process called beta-oxidation breaks down the fatty-CoA, 2 carbons at a time, to make acetyl CoA.
    • β-oxidation produces acetyl CoA and also FADH2 and NADH.
    • The acetyl CoA feeds into the Krebs cycle, and the FADH2 and NADH feed into the ETC.
    • On a per gram basis, fats give more energy than any other food source.
  • Protein metabolism
    • Proteins are broken down into amino acids by peptidases.
    • The nitrogen in the amino acid is converted to urea (for desert animals, birds and reptiles, it is uric acid).
    • The carbon in the amino acid is converted to pyruvate or acetyl-CoA, (or other metabolical intermediates such as oxaloacetate), depending on what amino acid it is.
    • The carbon products from amino acid metabolism can either feed into the Krebs cycle, or be the starting material for gluconeogenesis.