Medical Biochem Week 9 - Metabolic Integration

Metabolic Integration

• Citric Acid Cycle (CAC)
• Oxidative phosphorylation
• Lipid metabolism

Glycolysis: Aerobic and Anaerobic

• Pyruvate formed by glycolysis is further metabolized in one of three ways.
  • Hypoxic or anaerobic condition
    • Fermentation of Pyruvate to lactate
      • Muscles and erythrocytes
    • Fermentation of Pyruvate to ethanol
      • Yeast
  • Aerobic conditions
    • Pyruvate to Energy+CO2+H2O through
      • Citric Acid Cycle (CAC), also known as Tricarboxylic Acid Cycle (TAC) and Krebs cycle

The Citric Acid Cycle (CAC)

• What happens to Pyruvate (3C) under Aerobic conditions?
• CAC is also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle

Cellular Respiration

• Process in which cells consume O2 and produce Energy + CO2 + H2O
• Provides more energy (ATP) from glucose than glycolysis
• Also captures energy stored in lipids and amino acids
• Used by animals, plants, and many microorganisms
• Occurs in three major stages:
  • acetyl CoA production
  • acetyl CoA oxidation
  • electron transfer and oxidative phosphorylation
• $\text{GLUCOSE} \rightarrow \text{Glycolysis} \rightarrow \text{Pyruvate}$
  • $\Delta G'\degree = -146 \text{ kJ/mol}$
• $\text{GLUCOSE} + 6 \text{ O}<em>2 \rightarrow 6 \text{ CO}</em>2 + 6 \text{ H}_2\text{O}$
  • $\Delta G'\degree = -2,840 \text{ kJ/mol}$

Revision: Energy Carriers

• Other nucleotides involved in energy in metabolic pathways.
• Coenzyme A (CoA) functions in acyl group transfer reactions
• Nicotinamide Adenine dinucleotide (NAD+) functions in hydride transfers
• Flavin adenine dinucleotide (FAD), the active form of vitamin B2 (riboflavin) functions in electron transfers
  *Example: Acyl group

Acetyl-CoA Production

• The formation of pyruvate in glycolysis links glycolysis (in cytoplasm) to the CAC (in mitochondria).
• Coenzyme A (CoA) functions in acyl group transfer reactions

Acetyl-CoA Production

• Pyruvate Dehydrogenase Complex (PDH) catalyses the conversion of pyruvate to acetyl CoA.
• The reaction is irreversible.
• Oxidative decarboxylation of pyruvate
• Requires..
  • Prosthetic groups.
    • TPP, lipoyllysine, FAD
  • Co-substrates.
    • NAD+ and CoA-SH
• First carbons of glucose to be fully oxidized
• Note: OIL: Oxidation is loss of electrons
• RIG: Reduction is gain of electrons

The Citric Acid Cycle (CAC)

• Acetyl CoA (2C) is metabolized through Citric Acid Cycle to yield energy
• FADH2, GTP, NADH
• Use O2 to Produces CO2 = “Cellular Respiration”

The Citric Acid Cycle (CAC)

• Step 1: C-C bond formation between acetate (2C) and oxaloacetate (4C) to make citrate (6C)
• Step 2: Isomerization via dehydration/rehydration
• Steps 3–4: Oxidative decarboxylations to give 2 NADH
• Step 5: Substrate-level phosphorylation to give GTP
• Step 6: Dehydrogenation to give FADH2
• Step 7: Hydration
• Step 8: Dehydrogenation to give NADH
• Produces: 2 CO2 3 NADH, 1 FADH2 1 GTP(ATP) per Acetyl CoA
• Note: 1 glucose → 2 pyruvate → 2 Acetyl Co A

The Citric Acid Cycle (CAC) - Step 1

• C-C bond formation between acetate (2C) and oxaloacetate (4C) to make citrate (6C)
• Condensation of acetyl-CoA and oxaloacetate
• Small and stead supply of oxaloacetate is formed directly from pyruvate
• The only reaction with C-C bond formation
• Highly thermodynamically favorable/irreversible
• Rate-limiting step of CAC
• Activity largely depends on [oxaloacetate]
• Pyruvate carboxylase

The Citric Acid Cycle (CAC) - Step 2

• Step 2: Isomerization via dehydration/rehydration
• Citrate, a tertiary alcohol, is a poor substrate for oxidation.
• Isocitrate, a secondary alcohol, is a good substrate for oxidation.
• Aconitase eliminate H2O from citrate to form a cis C=C bond
• Addition of H2O to cis-aconitate is stereospecific.
• Thermodynamically unfavorable/reversible
• Product concentration kept low to pull forward

The Citric Acid Cycle (CAC) - Steps 3-4

• Steps 3–4: Oxidative decarboxylations to give 2 NADH
• Isozymes are specific for NADP+ (cytosolic) or NAD+ (mitochondrial).
• Decarboxylation releases CO2.
• Highly favorable/irreversible

The Citric Acid Cycle (CAC) - Steps 3-4 (cont.)

• Steps 3–4: Oxidative decarboxylations to give 2 NADH
• Succinyl-CoA is another higher-energy thioester bond.
• Highly thermodynamically favorable/irreversible
• regulated by product inhibition
• Last oxidative decarboxylation

The Citric Acid Cycle (CAC) - Step 5

• Step 5: Substrate-level phosphorylation to give GTP
• Substrate-level phosphorylation
• Produces GTP, which can be converted to ATP
• The energy of thioester allows for incorporation of inorganic phosphate.
• Goes through a phospho-enzyme intermediate
• Slightly thermodynamically favorable/reversible
• product concentration kept low to pull forward

The Citric Acid Cycle (CAC) - Step 6

• Step 6: Dehydrogenation to give FADH2
• FAD accepts Hydrogen from Succinate
• Reduction of the alkane (C-C) to alkene (C=C) via FADH2.
• Succinate Dehydrogenase is bound to mitochondrial inner membrane
• acts as Complex II in the electron-transport chain
• Near equilibrium/reversible, product concentration kept low to pull forward

The Citric Acid Cycle (CAC) - Step 7

• Step 7: Hydration
• Stereospecific
• OH− adds to fumarate… then H+ adds to the carbanion.
• Addition of water is always trans and forms L-malate.
• Slightly thermodynamically favorable/reversible
• product concentration kept low to pull reaction forward

The Citric Acid Cycle (CAC) - Step 8

• Step 8: Dehydrogenation to give NADH
• Regenerates oxaloacetate for citrate synthase
• Highly thermodynamically UNfavorable/reversible
• oxaloacetate concentration kept VERY low by citrate synthase - pulls the reaction forward

The Citric Acid Cycle (CAC) - Net Result

• Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O → 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
• Net oxidation of two carbons to CO2
• equivalent to two carbons of acetyl-CoA
• but NOT the exact same carbons
• Energy captured by electron transfer to NADH and FADH2
• Generates 1 GTP, which can be converted to ATP
• Completion of cycle

Regulation of the Citric Acid Cycle

• Controlled by:
  • Allosteric modulators (Enzyme regulation)
    • Thermodynamically favorable and irreversible steps
    • Binding of modulator to enzyme causes conformational change that changes the activity of the enzyme.
    • Can be inhibitory or stimulatory
      • NB. Not to be confused with uncompetitive and mixed inhibitors – these do not necessarily mediate conformational changes

Regulation of the Citric Acid Cycle (cont.)

• Controlled by:
  • Allosteric modulators (Enzyme regulation)
    • Thermodynamically favorable and irreversible steps
    • Citrate synthase - inhibited by citrate and also ATP
    • Isocitrate dehydrogenase – inhibited by NADH and ATP but activated by ADP & NAD+
    • α-ketoglutarate dehydrogenase is inhibited by NADH and succinyl CoA
    • Also Pyruvate dehydrogenase complex inhibited by NADH and ATP but also controlled by phosphorylation/ dephosphorylation by kinases and phosphatases and acetyl CoA

Regulation of the Citric Acid Cycle (cont.)

• Controlled by:
  • Allosteric modulators (Enzyme regulation)
    • Thermodynamically favorable and irreversible steps
  • General regulatory mechanism
    • activated by substrate availability
    • inhibited by product accumulation
    • Overall products of the pathway are NADH and ATP.
      • affect all regulated enzymes in the cycle
      • inhibitors: NADH and ATP
      • activators: NAD+ and AMP

The Citric Acid Cycle (CAC) - Significance

• The citric acid cycle (CAC) is:
  • Amphibolic (both catabolic and anabolic)
  • The cycle is involved in the aerobic catabolism of carbohydrates, lipids and amino acids
  • Intermediates of the cycle are starting points for many biosynthetic reactions
  • Enzymes of the cycle are in the mitochondria (eukaryotes) or the cytosol of bacteria
• Metabolic Pathways in cell are integrated and regulated to prevent waste

Oxidative Phosphorylation

• Energy of oxidation drives synthesis of ATP
• How is it achieved? - Not by another high- energy phosphate carrier
• ADP + Pi → ATP
  • highly thermodynamically unfavorable and requires energy
• Flow of protons (H+) down the electrochemical gradient generates ATP

Oxidative Phosphorylation (cont.)

• Energy of oxidation drives synthesis of ATP
• FADH2 → FAD + 2e- + 2H+
• NADH → NAD+ + 2e- + H+
• ATP → ADP + Pi

Oxidative Phosphorylation - Proton Gradient

• The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions.
  • plasma membrane in bacteria
  • inner membrane in mitochondria
  • thylakoid membrane in chloroplasts
• The membrane must contain proteins that couple the “downhill” flow of electrons in the electron-transfer chain with the “uphill” flow of protons across the membrane.
• The membrane must contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP.

Mitochondria

• Double membrane leads to four distinct compartments:
  • Outer membrane:
    • relatively porous membrane; allows passage of metabolites
  • Intermembrane space (IMS):
    • similar environment to cytosol
    • higher proton (H+) concentration (lower pH)
  • Inner membrane
    • relatively impermeable, with proton gradient across it
    • location of electron transport chain complexes
    • Convolutions called cristae serve to increase the surface area.
  • Matrix
    • location of the citric acid cycle and parts of lipid and amino acid metabolism
    • lower proton concentration (higher pH)

Electron Transport Chain

• Reduced substrate (fuel) donates e-
• Electron carriers (respiratory chain) pump H+ out as electrons flow to O2.

Electron transport chain - Complex I

• Complex I: NADH: Ubiquinone (Coenzyme Q) Oxidoreductase
• NADH binding site in the matrix side
• Noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH.
• Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site.
• Transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of protons from the matrix (N) to the intermembrane space (P).

Electron transport chain - Coenzyme Q

• Coenzyme Q (Ubiquinone) or Coenzyme Q is a mobile electron carrier transporting electrons from Complexes I and II to Complex III.

Electron transport chain - Complex I (cont.)

• Experiments suggest that about four protons are transported per one NADH.
• $\text{NADH} + \text{Q} + 5\text{H}^+<em>N = \text{NAD}^+ + \text{QH}</em>2 + 4 \text{H}^+_P$
• Protons are transported by proton wires.
  • a series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another
• Reduced coenzyme Q picks up two protons.

Electron transport chain - Complex II

• Complex II: Succinate Dehydrogenase
• FAD accepts two electrons from succinate.
• Electrons are passed, one at a time, via iron- sulfur centers to ubiquinone, which becomes reduced QH2.
• Does not transport protons
• Succinate dehydrogenase is a single enzyme with dual roles:
  • convert succinate to fumarate in the citric acid cycle
  • capture and donate electrons in the electron transport chain

Electron transport chain - Complex III

• Complex III: Ubiquinone:Cytochrome c Oxidoreductase
• Dimer of identical monomers, each with 11 different subunits
• Functional cores are
  • Cytochrome b and c1 with hemes
  • Rieske Iron-Sulphur proteins
• Uses two electrons from QH2 to reduce two molecules of cytochrome c
• Cytochrome c is a mobile electron carrier
• Clearance of electrons from the reduced quinones via the Q-cycle results in translocation of four additional protons (H+) to the intermembrane space.

Electron transport chain - Complex III (cont.)

• Net equation: $\text{QH}_2 + 2 \text{ cyt c (oxidized)} + 2\text{H}^+ \rightarrow \text{Q} + 2 \text{ cyt c (reduced)} + 4\text{H}^+$

Electron transport chain - Complex IV

• Complex IV: Cytochrome Oxidase
• Mammalian cytochrome oxidase is a membrane protein with 13 subunits.
• Contains two heme groups: a and a3
• Contains copper ions
  • CuA: two ions that accept electrons from cyt c
  • CuB: bonded to heme a3, forming a binuclear center that transfers four electrons to oxygen
• Four electrons are used to reduce one oxygen molecule into two water molecules.
• Four protons (H+) are picked up from the matrix in this process.
• Four additional protons (H+) are passed from the matrix to the intermembrane space.

Electron transport chain - Summary

• Complexes I, III, and IV pump protons from the matrix to the intermembrane space, creating an electrochemical gradient.

ATP Synthase Complex

• ATP synthase uses electrochemical potential to synthesize ATP.

ATP Synthase Complex - Structure

• Contains two functional units:
  • F0
    • Integral membrane complex
    • Proton translocation causes a rotation of the F0 subunit and the central shaft g.
  • F1
    • Soluble complex in the matrix
    • Hexamer arranged in three αβ dimers
    • Three different conformations:
      • open: empty
      • loose: binding ADP and Pi
      • tight: catalyzes ATP formation
    • Rotation causes a conformational change within all the three pairs.
    • One of three pairs promotes condensation of ADP and Pi into ATP

Metabolism of Lipids

• Produce lots of ATP
• Revision slide: Glycolysis produces ATP and Pyruvate (3C)
• Fatty acid breakdown → Pyruvate produce Acetyl CoA
• Acetyl CoA Used in CAC cycle, then Oxidative phosphorylation to

Metabolism of Lipids - Overview

• β-oxidation: oxidation of long chain fatty acids with the production of ATP
• Prokaryotes: Occurs in cytosol
• Eukaryotes: Occurs in mitochondrial matrix
• Fatty acids are converted to acyl CoA derivatives, then metabolized by removal of 2 carbon acetyl CoA units from the end of the chain (per cycle)
• Inner mitochondrial membrane is not permeable to longchain acyl CoA derivatives
• They are transported into mitochondria as carnitine derivatives by a transporter acyl carnitine translocase

Metabolism of Lipids - Stages

• Stage 1: β Oxidation ( FA → Acetyl CoA)
• Stage 2: Citric Acid Cycle
• Stage 3: Electron Transfer Chain

Metabolism of Lipids - Beta Oxidation Steps

• 4 Steps
  • Oxidation
  • Hydration
  • Oxidation
  • Thiolysis

Metabolism of Lipids - Activation of Fatty Acid

• Fatty acid + CoA → Fatty acyl-CoA
• Palmitate 16C Fatty acid + Fatty Acyl–CoA (Fatty acid with Coenzyme A attached)
• Activation of fatty acid (requires ATP)
• Fatty Acyl-CoA molecules - shuttled into the mitochondria via the Carnitine shuttle (acyl carnitine translocase)

Metabolism of Lipids - Four β-Oxidation Steps

• Oxidation (=dehydrogenation) – i.e removal of 2 Hydrogens. Creates a double bond.
• Removed Hs transferred to FAD→ FADH2

Metabolism of Lipids - Four β-Oxidation Steps (cont.)

• Hydration – addition of water

Metabolism of Lipids - Four β-Oxidation Steps (cont.)

• Oxidation (=dehydrogenation) – i.e removal of 2 Hydrogens. Creates a double bond.
• Removed Hs transferred to NAD+→ NADH + H+

Metabolism of Lipids - Four β-Oxidation Steps (cont.)

• Thiolysis – Acetyl CoA (2C) splits off from fatty acid

Metabolism of Lipids - Spiral Catabolic Pathway

• With each cycle the fatty acid gets shortened by 2C
• Shortened fatty acid re-enters the pathway, and gets shorter and shorter – spiral catabolic pathway.
• Acetyl Co-A, FADH2 and NADH (Hydrogens and electrons) enters CAC and electron transfer chain

Metabolism of Lipids - Energy Production

• Energy production in Palmitate:
• A 16 carbon FA (palmitate) undergoes 7 passes through the sequence - losing 2C each cycle.
• One round β-oxidation produces:
  • 1 NADH (3ATP), 1 FADH2 (2ATP) and 1 acetyl CoA (12 ATP in CAC) = 17ATP
• C16 Palmitate undergoes 7 rounds of β-oxidation (produces 8 acetyl CoA in total)
  • 7 NADH (21 ATP), 7 FADH2 (14 ATP) and 8 acetyl CoA (96 ATP in CAC)
  • -2 ATP (activation of palmitate) = 129 ATP
• The rate of FA oxidation is controlled by availability of FA in the blood arising from breakdown of triacylglycerols

Metabolism of Lipids - Fatty Acid Synthesis

• Involves the condensation of 2 carbon units (acetyl CoA) to form long hydrocarbons in a series of reactions
• Not the reverse of β-oxidation
  • Condensation
  • Reduction
  • Dehydration
  • Reduction
• Carried out on fatty acid synthase complex using NADPH as reductant
• Occurs in the cytosol (prokaryotes and eukaryotes)
• 4 Steps in β-oxidation
  • Oxidation
  • Hydration
  • Oxidation
  • Thiolysis

Metabolism of Lipids - Regulation of Fatty Acid Synthesis

• FA synthesis takes place when carbohydrate and energy are plentiful and fatty acids are scarce
• Synthesised FA then assembled into Triacylglycerols for storage
• Key enzyme is acetyl CoA carboxylase, regulated by covalent modifications (by a kinase)Regulation of Fatty acid synthesis