Wk9RG
Friday, March 1, 2019 9:03 PM
Citric Acid Cycle (CAC): Regulation
• Metabolites: usually describes the intermediates in the CAC.
Week 9 • Regulation occurs @ 3 levels:
○ The transport of pyruvate into the mitochondria via mitochondrial-pyruvate carrier (MPC)
○ The conversion of pyruvate to acetyl-CoA (PDH complex rxn)
○ The entry of acetyl-CoA into the cycle (citrate synthase rxn)
○ Isocitrate dehydrogenase- and a-ketoglutarate dehydrogenase- rxns
• Production of acetyl-CoA by the PDH complex is regulated by allosteric and covalent mechanisms:
• PDH complex is turned on when:
○ Low [acetate] flows into the CAC -> AMP, CoA, and NAD+ accumulates
○ Energy demands are high, cell requires greater [acetyl-CoA] for CAC
○ Basically: when [ATP/ADP], [NADH/NAD+], and [acetyl-CoA/CoA] ratios decrease -> pyruvate oxidation!
(Fig 16.19)
○ However, when energy is available (via fatty acids) -> allosteric inhibition of pyruvate oxidation!!
• PDH complex is inhibited by reversible phosphorylation of Ser residue on E1:
○ Pyruvate dehydrogenase kinase phosphorylates and inactivates E1.
○ Specific phosphoprotein phosphatase dephosphorylates and activates E1.
• High [ATP] -> activates PDH kinase -> phosphorylates E1 -> PDH complex is inactivated.
○ FUN FACT: dichloroacetate inhibits PDH kinase in lab -> pyruvate oxidation!
• The CAC is regulated at it's 3 exergonic (first, rate-limiting) steps:
○ Steps catalyzed by citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase.
○ Ex. ATP inhibits citrate synthase, yet ADP allosterically activates this enzyme.
• *In general, a rxn is regulated by [S], inhibition by accumulating products, and allosteric inhibition of enzymes.
• Pathway geared towards rate that provides optimal levels of [ATP] and [NADH].
• Pathway RECAP: Glycolysis (cytosol) produces glucose -> pyruvate (transported into mitochondria)-> acetyl-CoA
(via PDH complex) -> enters Citric Acid Cycle!
• Substrate channeling thru multienzyme complexes may occur in the CAC.
• Why are cell contents broken into a crude extract during purification?
○ Buffer reduces the concentration of enzymes present -> favors dissociation of multienzyme complexes.
• Metabolons: multienzyme complexes that help it's products reach the next enzyme in the pathway.
○ These complexes controls substrate's solubility in the inner mitochondrial membrane -> decide when
substrate can leave enzyme complex (channel their substrates).
• Some mutations in CAC enzymes lead to cancer.
• Inhibition of these enzymes could interfere w/ normal cell division -> tumors.
• Ex. Fumerase mutation: fumarate (substrate) accumulates -> induces TF HIF-1a -> upregulation of genes
normally regulated by HIF-1a.
ETC, PART 2: ATP Synthase
• In the Chemiosmotic Model, oxidation and phosphorylation are "obligately coupled."
• Chemiosmotic model: the proton-motive force drives ATP synthesis as protons flow passively back into the
matrix thru ATP synthase. (Fig 19.19)
○ Proton-motive force: proton concentration difference (against gradient) + charge separation across the
inner mitochondrial membrane.
• -> Electron transfer and ATP synthesis rxns cannot occur w/o the other.
• Experimental evidence found on p. 730. Once succinate (substrate) is added to ADP and P in a medium ->
respiration begins immediately and ATP is synthesized. Addn of CN- (inhibitor) blocks e- transfer b/w complex IV
and O2. -> inhibits BOTH rxns.
○ Vice versa, when ADP and P are added to succinate.
• Makes sense: ATP synthase is blocked -> proton builds up in intermembrane space of mitochondria -> proton
gradient ≥ electron transfer from NADH to O2 -> e- flow must stop to accommodate H+ gradient.
• EXCEPTION Certain conditions and reagents can uncouple oxidation from phosphorylation:
○ Detergent, or physical shear: e- transfer still occurs, but no ATP synthesis
○ Chemical uncouplers: weak, hydrophobic acids that can pick up and drop off protons into matrix
(deprotonated anionic form more water-soluble) -> created a proton gradient w/o electron transfer via
oxidation. REREAD IN TEXTBOOK
, • More experimental evidence on p. 731. Researchers able to impose a proton-motive force on inner membrane
w/o an oxidizable substrate -> ATP was synthesized!
○ pH 9 to 7 (more H+ in intermembrane space); valinomycin was added (uncoupler); no added K+ (no
oxidative substrate).
• ATP synthase has two domains: F0 and F1.
• F1: peripheral membrane protein
• F0: integral (inside) to the membrane; oligomycin-sensitive.
• F1 plugs F0's proton pore, allowing proton gradient to occur and e- transfer to couple w/ ATP synthesis.
• ATP is stabilized relative to ADP on F1 surface.
• **ATP synthase binds ATP more tightly, releasing enough energy to counterbalance cost of ATP production. ->
ATP stabilizes relative to ADP + P.
• *ATP synthase has high affinity for ATP, and low affinity for ADP.
• Proton gradient drives release of ATP from the enzyme surface.
• Proton-motive force provides free energy required to release newly-synthesized ATP.
• ***HIGH affinity for ATP (low Km) -> synthase binds more tightly w/ ATP -> release of ATP from synthase
enzyme is largest energy barrier in rxn coordination diagram.
○ Despite low affinity for ADP, ADP phosphorylation is reversible (Keq = 1, delta G = 0).
• Each B subunit (alternating subunit of F1) of ATP synthase can assume 3 different conformations.
• Association of F0 √ subunit w/ one of three subunits -> 3 B subunits have conformations that differ, despite
having identical a.a. sequences.
• Overall, top, and side view of B subunits on p. 734.
• B (beta) subunit conformations:
○ B-ADP: binding site filled w/ ADP. Binds ADP to Pi. ->
○ B-ATP: binding site filled w/ App(NH)p (ATP analog that cannot be hydrolyzed.) ->
○ B-empty: binding site is empty. Has LOW affinity for ATP -> ATP leaves enzyme surface.
• No subunit can have the same conformation during a cycle.
• a and B subunits are not rotating, just the shaft! Proton motive force only changes conformation.
• Rotation catalysis is key to the binding-change mechanism for ATP synthesis.
• Binding-change model: √ subunit rotates in one direction when F0F1 (ATP synthase) is synthesizing ATP and
another direction when hydrolyzing ATP.
• *√ subunit acts as shaft of F0, also rotating ring of c subunits (8-15c, depending on organism).
• Proton flow thru F0 complex -> rotary motion:
1. Proton enters P side (from intermembrane space) half-channel.
2. Protonates the Asp of the next c subunit.
3. Displaces Arg associated w/ Asp.
4. Arg rotates, and displaces proton of Asp from next c subunit. H+ exits N side (into matrix) half-channel.
○ c ring rotates so Arg can return to P side half-channel!
• Chemiosmotic coupling allows nonintegral stoichiometries of O2 consumption and ATP synthesis. p.
738
• The proton-motive force also energizes active transport.
• Proton gradient can also drive transport processes:
• Adenine nucleotide translocase: integral membrane channel that transports ADP3- into the matrix in exchange
for ATP4- transported outwards.
○ 4 negative charges out for every 3 moved in is favored by the transmembrane electrochemical gradient.
○ Electrochemical gradient: gives matrix a net negative charge.
○ Fig 19-30 on p. 738.
• Phosphate translocase: transports H2PO4- and H+ into the matrix.
○ Also favored by proton gradient; however, the proton movement consumes some energy of e- transfer.
○ Intermembrane space has higher proton (H+) concentration!
ETC, PART 1: Electron Transport Chain (oxidation phosphorylation)
• Oxidative phosphorylation: in eukaryotes, it is ATP synthesis in the mitochondria. Chemiosmotic
mechanism: Fig. 19-1, pg. 711
1. EXERGONIC reduced substrate donates e- thru ETC, to a final e- acceptor w/ large reduction
potential (O2).
2. ENDERGONIC as e- flow to O2, electron carriers pump H+ out. ETC goes against
concentration gradient.
3. Energy of e- flow is stored as electrochemical potential. Transmembrane flow of H+ back down
their concentration gradient.
4. ATP synthase uses electrochemical potential to synthesize ATP (phosphorylates ADP).
Friday, March 1, 2019 9:03 PM
Citric Acid Cycle (CAC): Regulation
• Metabolites: usually describes the intermediates in the CAC.
Week 9 • Regulation occurs @ 3 levels:
○ The transport of pyruvate into the mitochondria via mitochondrial-pyruvate carrier (MPC)
○ The conversion of pyruvate to acetyl-CoA (PDH complex rxn)
○ The entry of acetyl-CoA into the cycle (citrate synthase rxn)
○ Isocitrate dehydrogenase- and a-ketoglutarate dehydrogenase- rxns
• Production of acetyl-CoA by the PDH complex is regulated by allosteric and covalent mechanisms:
• PDH complex is turned on when:
○ Low [acetate] flows into the CAC -> AMP, CoA, and NAD+ accumulates
○ Energy demands are high, cell requires greater [acetyl-CoA] for CAC
○ Basically: when [ATP/ADP], [NADH/NAD+], and [acetyl-CoA/CoA] ratios decrease -> pyruvate oxidation!
(Fig 16.19)
○ However, when energy is available (via fatty acids) -> allosteric inhibition of pyruvate oxidation!!
• PDH complex is inhibited by reversible phosphorylation of Ser residue on E1:
○ Pyruvate dehydrogenase kinase phosphorylates and inactivates E1.
○ Specific phosphoprotein phosphatase dephosphorylates and activates E1.
• High [ATP] -> activates PDH kinase -> phosphorylates E1 -> PDH complex is inactivated.
○ FUN FACT: dichloroacetate inhibits PDH kinase in lab -> pyruvate oxidation!
• The CAC is regulated at it's 3 exergonic (first, rate-limiting) steps:
○ Steps catalyzed by citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase.
○ Ex. ATP inhibits citrate synthase, yet ADP allosterically activates this enzyme.
• *In general, a rxn is regulated by [S], inhibition by accumulating products, and allosteric inhibition of enzymes.
• Pathway geared towards rate that provides optimal levels of [ATP] and [NADH].
• Pathway RECAP: Glycolysis (cytosol) produces glucose -> pyruvate (transported into mitochondria)-> acetyl-CoA
(via PDH complex) -> enters Citric Acid Cycle!
• Substrate channeling thru multienzyme complexes may occur in the CAC.
• Why are cell contents broken into a crude extract during purification?
○ Buffer reduces the concentration of enzymes present -> favors dissociation of multienzyme complexes.
• Metabolons: multienzyme complexes that help it's products reach the next enzyme in the pathway.
○ These complexes controls substrate's solubility in the inner mitochondrial membrane -> decide when
substrate can leave enzyme complex (channel their substrates).
• Some mutations in CAC enzymes lead to cancer.
• Inhibition of these enzymes could interfere w/ normal cell division -> tumors.
• Ex. Fumerase mutation: fumarate (substrate) accumulates -> induces TF HIF-1a -> upregulation of genes
normally regulated by HIF-1a.
ETC, PART 2: ATP Synthase
• In the Chemiosmotic Model, oxidation and phosphorylation are "obligately coupled."
• Chemiosmotic model: the proton-motive force drives ATP synthesis as protons flow passively back into the
matrix thru ATP synthase. (Fig 19.19)
○ Proton-motive force: proton concentration difference (against gradient) + charge separation across the
inner mitochondrial membrane.
• -> Electron transfer and ATP synthesis rxns cannot occur w/o the other.
• Experimental evidence found on p. 730. Once succinate (substrate) is added to ADP and P in a medium ->
respiration begins immediately and ATP is synthesized. Addn of CN- (inhibitor) blocks e- transfer b/w complex IV
and O2. -> inhibits BOTH rxns.
○ Vice versa, when ADP and P are added to succinate.
• Makes sense: ATP synthase is blocked -> proton builds up in intermembrane space of mitochondria -> proton
gradient ≥ electron transfer from NADH to O2 -> e- flow must stop to accommodate H+ gradient.
• EXCEPTION Certain conditions and reagents can uncouple oxidation from phosphorylation:
○ Detergent, or physical shear: e- transfer still occurs, but no ATP synthesis
○ Chemical uncouplers: weak, hydrophobic acids that can pick up and drop off protons into matrix
(deprotonated anionic form more water-soluble) -> created a proton gradient w/o electron transfer via
oxidation. REREAD IN TEXTBOOK
, • More experimental evidence on p. 731. Researchers able to impose a proton-motive force on inner membrane
w/o an oxidizable substrate -> ATP was synthesized!
○ pH 9 to 7 (more H+ in intermembrane space); valinomycin was added (uncoupler); no added K+ (no
oxidative substrate).
• ATP synthase has two domains: F0 and F1.
• F1: peripheral membrane protein
• F0: integral (inside) to the membrane; oligomycin-sensitive.
• F1 plugs F0's proton pore, allowing proton gradient to occur and e- transfer to couple w/ ATP synthesis.
• ATP is stabilized relative to ADP on F1 surface.
• **ATP synthase binds ATP more tightly, releasing enough energy to counterbalance cost of ATP production. ->
ATP stabilizes relative to ADP + P.
• *ATP synthase has high affinity for ATP, and low affinity for ADP.
• Proton gradient drives release of ATP from the enzyme surface.
• Proton-motive force provides free energy required to release newly-synthesized ATP.
• ***HIGH affinity for ATP (low Km) -> synthase binds more tightly w/ ATP -> release of ATP from synthase
enzyme is largest energy barrier in rxn coordination diagram.
○ Despite low affinity for ADP, ADP phosphorylation is reversible (Keq = 1, delta G = 0).
• Each B subunit (alternating subunit of F1) of ATP synthase can assume 3 different conformations.
• Association of F0 √ subunit w/ one of three subunits -> 3 B subunits have conformations that differ, despite
having identical a.a. sequences.
• Overall, top, and side view of B subunits on p. 734.
• B (beta) subunit conformations:
○ B-ADP: binding site filled w/ ADP. Binds ADP to Pi. ->
○ B-ATP: binding site filled w/ App(NH)p (ATP analog that cannot be hydrolyzed.) ->
○ B-empty: binding site is empty. Has LOW affinity for ATP -> ATP leaves enzyme surface.
• No subunit can have the same conformation during a cycle.
• a and B subunits are not rotating, just the shaft! Proton motive force only changes conformation.
• Rotation catalysis is key to the binding-change mechanism for ATP synthesis.
• Binding-change model: √ subunit rotates in one direction when F0F1 (ATP synthase) is synthesizing ATP and
another direction when hydrolyzing ATP.
• *√ subunit acts as shaft of F0, also rotating ring of c subunits (8-15c, depending on organism).
• Proton flow thru F0 complex -> rotary motion:
1. Proton enters P side (from intermembrane space) half-channel.
2. Protonates the Asp of the next c subunit.
3. Displaces Arg associated w/ Asp.
4. Arg rotates, and displaces proton of Asp from next c subunit. H+ exits N side (into matrix) half-channel.
○ c ring rotates so Arg can return to P side half-channel!
• Chemiosmotic coupling allows nonintegral stoichiometries of O2 consumption and ATP synthesis. p.
738
• The proton-motive force also energizes active transport.
• Proton gradient can also drive transport processes:
• Adenine nucleotide translocase: integral membrane channel that transports ADP3- into the matrix in exchange
for ATP4- transported outwards.
○ 4 negative charges out for every 3 moved in is favored by the transmembrane electrochemical gradient.
○ Electrochemical gradient: gives matrix a net negative charge.
○ Fig 19-30 on p. 738.
• Phosphate translocase: transports H2PO4- and H+ into the matrix.
○ Also favored by proton gradient; however, the proton movement consumes some energy of e- transfer.
○ Intermembrane space has higher proton (H+) concentration!
ETC, PART 1: Electron Transport Chain (oxidation phosphorylation)
• Oxidative phosphorylation: in eukaryotes, it is ATP synthesis in the mitochondria. Chemiosmotic
mechanism: Fig. 19-1, pg. 711
1. EXERGONIC reduced substrate donates e- thru ETC, to a final e- acceptor w/ large reduction
potential (O2).
2. ENDERGONIC as e- flow to O2, electron carriers pump H+ out. ETC goes against
concentration gradient.
3. Energy of e- flow is stored as electrochemical potential. Transmembrane flow of H+ back down
their concentration gradient.
4. ATP synthase uses electrochemical potential to synthesize ATP (phosphorylates ADP).
