lec 14 - cell metabolism and beyond (zong)

metabolism

• the breakdown and synthesis of macromolecules in living cells

• macromolecules include:

  • lipids

  • nucleic acid

  • carbs

  • proteins

catabolism and anabolism

• catabolism = the degradative phase of metabolism → releases energy

• anabolism = the building phase of metabolism → requires energy

• connected by carrier molecules that store energy and electrons

  • ATP

  • NADH

  • NADPH

  • FADH2

anabolism

• precursor molecules

  • AA

  • sugars

  • fatty acids

  • nitrogenous bases

• goes through anabolism which involves carrier molecules and chemical energy

  • ATP → ADP + HPO₄^2-

  • NADPH → NAD+

  • NADH → NADP+

  • FADH₂ → FAD

• results in cell macromolecules

  • proteins

  • polysaccharides

  • lipids

  • nucleic acids

catabolism

• energy-containing nutrients

  • carbs

  • fats

  • proteins

• goes through catabolism which involves energy being released as the macromolecules are broken down and that energy is used to regenerate carrier molecules

  • ADP + HPO₄^2- → ATP

  • NAD+ → NADH

  • NADP+ → NADPH

  • FAD → FADH₂

• results in energy-depleted end products

  • CO2

  • H2O

  • NH3

catabolic pathways vs anabolic pathways

catabolic pathways converge and anabolic pathways diverge

• complex molecules are broken down into simple building blocks which can then be used to build complex molecules

diagram

• for catabolism

  • multiple diverse molecules (starch, glycogen, sucrose) are broken down into common intermediate acetyl-coA → enters TCA cycle for further oxidation to CO2, oxaloacetate (allows it to keep running)

• for anabolism

  • from common intermediate, acetyl-coA many biosynthetic pathways:

    • fatty acids → triacylglycerols

    • mevalonate → cholesterol → bile acids, steroid hormones

    • isopentenyl-pyrophosphate → carotenoids, vit K, rubber

catabolism: energy production

combustion rxn

• organic compound + O₂ → energy (heat and light) + CO₂ + H₂O

  • all energy is released quickly and uncontrollably → NOT usable by cells

cell respiration

• organic compound (food) + O₂ → ATP + CO₂ + H₂O

  • energy is released gradually and stored in ATP which powers biological functions

ATP

• adenosine triphosphate → “energy cash” in the cell

  • organic molecule containing high energy phosphate bonds

  • structure

    • 3 phosphate groups → each is negatively charged

      • breaking one of these bonds releases energy used to fuel cellular processes

    • ribose sugar

    • adenine base

    • sugar + base = adenosine

how do we get energy from ATP?

• by breaking the high energy bonds between the last 2 phosphates in ATP

• in humans, ~60% energy generated by ATP hydrolysis is to produce heat

electron carrier for energy storage: NADH, FADH₂

• the electrons stored in NADH and FADH₂ = high energy → can be 'cashed’ to product ATP

• NADH

  • NAD+ + 2e- + H+ = NADH (reduced form that holds energy)

• FADH₂

  • FAD + 2e- + 2H+ = FADH₂

• analogy

  • ATP = energy ‘cash’

  • NADH and FADH₂ = energy ‘bonds’ (savings that can be converted to cash)

bioenergy comes from redox reaction: transfer of electrons

• electron donor = being oxidized

  • THINK: OIL RIG

    • oil = oxidation is loss of electrons → donating electrons

• electron receiver = being reduced

  • THINK: opposite of reduction is gain → gain of electrons

• energy is released when electrons are transferred from high energy state to low energy state; this energy can be harnessed by pumping H+ to generate proton gradient then producing ATP

• examples

  • Mg + Cl₂ → Mg²⁺ + 2Cl^-

    • Mg is oxidized → loses 2 e-

    • Cl is reduced → each Cl atom gains 1 e-

glucose metabolism

• steps

  • glucose enters the cell from the blood via a glucose transporter

  • in the cell, hexokinase converts glucose into glucose-β-phosphate

  • glucose-β-phosphate is broken down into pyruvate which produces 2 ATP and H+

    • H+ leaves cell through sodium-hydrogen exchanger and into blood

  • pyruvate has 2 pathways

    • aerobic pathway (with O₂)

      • pyruvate enters the mitochondria and undergoes: pyruvate oxidation, TCA, electron transport chain → final output = 36 ATP + H+

      • HCO3- is a byproduct → goes through anion exchanger → into blood

    • anaerobic pathway (without O₂)

      • pyruvate → lactate → regenerates NAD+, allowing glycolysis to continue → lactate transporter out of cell using mono-carboxylate transporter → into blood

glucose transporters

general note

• lower kM = higher affinity

• bloog glucose level roughly 3.9-8.3 mM

GLUT1

• tissue location

  • all other mammalian tissue

• Km

  • 1 mM

• responsive to insulin

  • NO

• comments

  • basal glucose uptake in erythrocytes and brain

GLUT2

• tissue location

  • liver

  • also pancreatic β cells

  • kidney cells

• Km

  • 15-20 mM

• responsive to insulin

  • NO

• comments

  • in liver, removes excess glucose from the blood

  • also used for export of glucose from liver

GLUT3

• tissue location

  • all other mammalian tissues

• Km

  • 1 mM

• responsive to insulin

  • NO

• comments

  • major glucose transporter in neurons

GLUT4

• tissue location

  • muscle and fat cells

• Km

  • 5 mM

• responsive to insulin

  • YES

• comments

  • expression in muscle plasma membrane increases with endurance training

  • you need insulin 4 GLUT 4 in your 4 limbs of muscle and fat

GLUT5

• tissue location

  • small intestine

• Km

  • n/a

• responsive to insulin

  • NO

• comments

  • primarily a fructose transporter

10 steps of glycolysis

• steps 1 and 3 = investment steps to prepare glucose to be split (use ATP)

• steps 7 and 10 = substrate level of phosphorylation to generate ATP

• prep phase = 1-5

  • priming glucose molecules with phosphate groups to make it easier to split → uses 2 ATP

• payoff phase = 6-10

  • extract energy

overview of glycolysis

1. oxidation and cleavage of glucose

2. in all cells

3. the hub of carbohydrate metabolism b/c virtually all carbohydrates can be converted to glucose

4. occurs in the cytosol

5. converts 6-C glucose → 3-C pyruvate

6. net products = 2 pyruvate, 2 NADH, 2 ATP

phase I: energy investment

• 6-C glucose phosphorylated using 2 ATP → fructose-1,6-biphosphate → splits into two 3-C molecules called glyceraldehyde 3-P

phase II: energy payoff

• glyceraldehyde-3-P → phosphate added from P_i (free floating phosphate present in cytosol) → 1,3-biphosphoglycerate → NAD+ converted to NADH + H+ and 2 ADP converted to 2 ATP → pyruvate

• 4 ATP produced but net 2 ATP b/c u consumed 2 in phase 1

feeder pathways

• all carbohydrates enter glycolysis

• in muscle, often via hexokinase

• lactose intolerance = lactase deficiency

  • not enough lactase available to convert lactose into D-galactose which then gets made into intermediates for glycolysis

• image shows how variety dietary carbs are funneled into glycolysis → all carbs must be converted into intermediates of glycolysis like G-6-phosphate to be metabolized for energy

fate of the products: pyruvate and NADH

glucose → glycolysis (10 successive reactions) → 2 pyruvate

• aerobic conditions: animal, plant, and many microbial cells under aerobic conditions

  • 2 pyruvate enters mitochondria → 2 CO2 is released as its converted to 2 acetyl-coA → acetyl coA enters the citric acid cycle (TCA) → produces 4 CO₂ + 4 H₂O

• anaerobic conditions: fermentation to lactate in vigorously contracting muscle, in erythrocytes, in some other cells and in some other micro-organisms

  • 2 pyruvate → 2 lactate

• hypoxic or anaerobic conditions: fermentation to ethanol in yeast

  • 2 pyruvate → 2 ethanol + 2 CO₂
    

fermentation in animals

• intense exercise → anaerobic metabolism and lactate production

  • muscle consume oxygen quickly, sometimes faster than its deliver → creates hypoxic or anaerobic conditions in muscle

• pyruvate + NADH + H+ → lactate + NAD+

  • through lactate dehydrogenase

  • regenerates NAD+ to keep glycolysis going (CANNOT make ATP without it)

  • allows short-term ATP production even WITHOUT oxygen

cori cycle

• lactate produced by anaerobic glycolysis in muscles gets transferred into the liver where lactate is used for glucose production then sent back to muscles

• purpose

  • facilitate muscle exertion

  • prevents lactate acidosis

  • main source of gluconeogenesis during fasting

• diagram

  • muscle

    • glucose goes through glycolysis and creates energy in muscle → generates pyruvate → lactate

  • liver

    • lactate goes to liver → becomes pyruvate → expends energy to create glucose in process called gluconeogenesis → glucose goes back to muscle so that it can continue glycolysis to create energy

fermentation in yeast

• example = beer

• pyruvate → acetaldehyde + CO₂

  • enzyme catalyzing rxn = pyruvate decarboxylase

  • cofactor = thiamine pyrophosphate (TPP) and Mg²⁺

• acetaldehyde → ethanol

  • enzyme= alcohol dehydrogenase

  • NADH + H → NAD+

    • uses this to reduce acetaldehyde

    • regenerates NAD+ to keep glycolysis running

      • NAD+ is coupled to ATP which is why its regeneration is important

liver cell - hepatocyte

fed state → hepatic glucose uptake and utilization

• insulin is released from pancreas in response to high blood glucose and binds to insulin receptor of hepatocyte and stimulates GK

• glucose is brought into cell via GLUT2 transporter → glucokinase (GK) phosphorylates → glucose-6-phosphate

  • → glycolysis → pyruvate → lipid synthesis

  • glycogen (storage form of glucose)

fasted state

• glucagon is secreted when blood glucose is low and binds to receptor of hepatocyte signals to glycogen

• glycogen → release glucose-6-phosphate → G6Pase converts glucose-6-phosphate into free glucose → transported through GLUT2 out into blood

skeletal muscle cell

rapidly contracting state (e.g. exercising)

• low ATP/ high AMP (signal of low energy)

• glucose is brought into cell via GLUT4 transporters → hexokinase converts glucose to glucose-6-phosphate → glycolysis → ATP

• also stored glycogen is broken down into G6P to further fuel glycolysis

• outcome = max ATP production to meet urgent energy demand of muscle contractions

resting state

• high ATP/low AMP

• glucose is brought into cell via GLUT4 transporters → converted to G6P → instead of fueling glycolysis, channeled towards glycogen synthesis (storage)

• → also has glycolysis but it slows down b/c cell already has enough ATP

• outcome = energy is conserved and glucose is stored as glycogen for future use

pyruvate enters mitochondria to be further oxidized

carbs get converted to glucose and then to pyruvate where it enters the mitochondria and goes thru TCA cycle

PDHC catalyzes the oxidative decarboxylation of pyruvate

• product of oxidative decarboxylation of pyruvate is acetyl coA

• irreversible rxn

  • produces NADH

  • releases the first free CO₂

  • thiamin pyrophosphate (TPP) = coenzyme

  • inhibited by arsenic

• pyruvate → acetyl coA

  • enzyme = pyruvate dehydrogenase complex

  • NAD+ → NADH

  • CoA-SH attaches to the acetyl group to form acetyl-coA

overview of the TCA cycle

• occurs in the mitochondria

• occurs in all organs except those that lack mitochondria

• aerobic pathway

main functions of the TCA (tri-carboxylic acid) cycle

• citric acid oxidizes the 2 carbons of acetyl coA → CO₂

• in the process of oxidation, high-energy electrons are captured in the form of NADH and FADH₂

• key function = harvest high-energy electrons from carbon fuels which can then be used by the electron transport chain (ETC) for oxidative phosphorylation

TCA cycle

• in step 1 of TCA cycle, 2 carbon acetyl CoA condenses with 4-carbon oxaloacetate to form 6-carbon citrate through citrate synthase

• although oxygen is NOT directly involved in any of the steps, TCA cycle is tightly coupled to the ETC and oxidative phosphorylation and is thus dependent on O₂

• 4 distinct classes of dehydrogenases in the TCA cycle = responsible for electron capture to generate NADH and FADH₂

  • isocitrate dehydrogenase

    • produce CO₂ → literally just take it off the molecule

  • α-ketoglutarate dehydrogenase complex

    • produce CO₂ → literally just take it off the molecule

  • succinate dehydrogenase

    • gives free electrons to FAD → FADH₂

    • forms double bond

  • malate dehydrogenase

    • gives free electrons to NAD → NADH

    • forms double bond

• 1 turn through TCA produces:

  • 3 NADH molecules

  • 1 FADH₂ molecules

  • 1 GTP molecule

  • releases 2 molecules of CO₂

cellular energy levels determine the rate of TCA cycle

• when cellular energy charge is high, TCA cycle is inhibited

  • high levels of NADH (NADH is used to make ATP in ETC) → slows down TCA cycle and PDH activity

• when cellular energy charge is low, TCA cycle is activated

  • lots of muscle activity → high Ca2+ levels → stimulates TCA cycle and PDH activity

TCA cycle intermediates can participate in both catabolic and anabolic rxns

• many of the intermediates of the TCA cycle participate in other metabolic reactions

• energy and metabolic needs of the cell dictate the direction of the rxns

• since intermediates of the TCA cycle can participate in both catabolic and anabolic reactions → reactions are described as anaplerotic

• example

  • citrate → fatty acids or steroids

  • alpha-ketoglutarate → glutamate → other AA → purines

anaplerosis

• chemical reactions that form intermediates of a metabolic pathway

• examples of such are found in the TCA cycle

• in the normal function of the TCA cycle for respiration, concentrations of TCA intermediates remain constant

• however, many biosynthetic reactions also use these molecules as substrate → in the context of the TCA cycle, anaplerosis = process of replenshing intermediates that have bene extracted for biosynthesis

energy yielded from the TCA cycle

• starting with acetyl-coA the final reaping of 1 TCA cycle is as follows

  • 3 NADH → 3 NAD+ → 9 ATP

  • FADH₂ → FAD → 2 ATP

  • GDP + P_i → GTP

  • in total = 12 ATP/acetyl coA oxidized

energy “cashing out” by oxidative phosphorylation in mitochondria

• TCA is in mitochondrial matrix

• oxidative phosphorylation = ETC + chemiosmosis

  • occurs in inner mitochondrial membrane

• you eject H+ into intermembrane space which then flows back into the mitochondrial matrix to generate ATP

oxidative phosphorylation

5 total complexes

• complex I = NADH dehydrogenase

  • NADH → NAD+, donates e- to FMN and Fe-S

• complex II = succinate dehydrogenase

  • feeds electrons via FADH₂

• complex III = cytochrome bc1 = cytochrome reductase

  • passes e- to Cyt C

• complex IV = cytochrome c oxidase

  • transfers e- to O₂ → forms H₂O

• complex V = ATP synthase

  • ADP + P_i → ATP

  • chemiosmosis

standard oxidation-reduction potential, E^’o

• the more positive the E’o value → higher affinity for electrons

• the standard oxidation-reduction potential is a measure of the affinity for a compound to accept electrons

• also called redox potential

• a redox pair with a higher Eo has a higher affinity for electrons than a redox pair with lower Eo

• redox pair with higher Eo will “take” electrons from a redox pair with a lower Eo

in the ETC, electrons from NADH and FADH₂ are transferred step wise to a series of electron acceptors

• electrons from NADH and FADH₂ move in a stepwise fashion through the ETC towards oxygen → passing to lower and lower energy states and releasing energy at each step

• enables cell to maximize the harvesting of energy from the ETC to drive ATP production

  • so without the chain it would be a huge, wasteful burst of energy but with ETC its small manageable steps where lots of ATP is made

• O₂ pulls electrons down the chain in an “energy-yielding tumble”

in oxidative phosphorylation, chemiosmosis couples ETC to ATP synthesis

• the energy stored in a H+ ion gradient across the inner mitochondrial membrane is used to synthesize ATP

• the H+ gradient is referred to as proton-motive force highlighting its capacity to do work

• some energy is lose as heat → plays role in maintaining body temp

• diagram

  • F₁ = in the mitochondrial matrix contains the catalytic activity

  • F_o = in the inner mitochondrial membrane contains the H+ pore

  • chemical potential inside matrix = alkaline

  • electrical potential inside matrix = negative

ATP synthesis is coupled to ETC

• cyanide = complex IV inhibitor → blocks electron transfer to oxygen which halts electron flow, H+ pumping, ATP production

ATP from complete oxidation of glucose

anaerobic

• glycolysis → 2 ATP

aerobic

• glycolysis → 1 NADH generates 3 ATP → 2 NADH so 6 ATP

• pyruvate → acetyl coA

  • 1 NADH generates 3 ATP → 2 NADH so 6 ATP

• acetyl coA → TCA cycle

  • 3 NADH → 9 ATP

  • 1 FADH₂ → 2 ATP

  • 1 GTP → 1 ATP

  • total 12 ATP but whole thing goes through twice because 2 pyruvate molecules so 2 acetyl coA

• total for aerobic = 36 ATP

breakdown of carbs, AA, and fatty acids

• sugars, fatty acids and AA can all be broken down to acetyl coA → all 3 can generate energy through the TCA cycle

• final reaction

  • Acetyl CoA + 3NAD+ + FAD + ADP + Pi + 2 H2O → 2 CO2 + 3 NADH + FADH2 + ATP + 2H+ + CoA