Biochem 6 Flashcards
1
Q
krebs cycle/tricarboxylic acid/citric acid
A
- one substrate- acetyl CoA
- one product- CO2
- its not get a way to oxidize acetyl CoA or make ATP
2
Q
only a small amount of energy available in glucose is captured in glycolysis
A
- complete oxidation only using the glycolytic path only generates pyruvate acid from glucose and a certain amount of free energy to make ATP
- complete oxidation of glucose (glycolysis and krebs) -> makes a lot more free energy and ATP
- amount per glucose is not the important part -> you can upregulate
- if the flux of the glycolytic pathway can be increased by 2 orders of magnitude -> doesnt matter how many ATP you can get per glucose (as long you have a good amount)
3
Q
in eukaryotes, stages 2 and 3 are localized to the mitochondria
A
- glycolysis occurs in the cytoplasm
- citric acid cycle occurs in the mitochondrial matrix
- mitochondrial matrix has high concentration of enzymes (soluble)
- oxidative phosphorylation occurs in the inner membrane
- there are proteins/enzymes in the inner membrane -> carriers for the ETC
- succinate dehydrogenase -> enzyme participates in the krebs cycle and ETC
- proteins in the inner membrane transport protons
- matrix is proton poor compared to cytosol (proton rich)
- proton movement from the cytosol to the matrix through the ATP synthase -> synthesis of ATP
4
Q
respiration: stage 2: acetyl-CoA oxidation
A
- krebs cycle
- generates more NADH, FADH2, and one GTP
- substrate level phosphorylation converts GDP -> GTP
- acetyl-CoA was synthesized from pyruvate
- amino acids (glutamic acid and glutamine) can also enter the krebs cycle -> important role when the cycle is serving to be the first step of gluconeogenesis
- remaining carbon atoms from carbohydrates, amino acids, and fatty acids are released during stage 2
5
Q
respiration: stage 3: oxidative phosphorylation
A
- generates the vast majority of ATP in well oxygenated well perfused tissues during catabolism
- inner membrane
6
Q
sequence of events in oxidative decarboxylation of pyruvate
A
- enzyme 1:
- step 1- decarboxylation of pyruvate to an active aldehyde
- step 2- oxidation of aldehyde to a carboxylic acid
- electrons reduce lipoamide and form a thioester
- enzyme 2:
- step 3- formation of acetyl-CoA (product 1)
- enzyme 3:
- step 4- reoxidation of the lipoamide cofactor
- step 5- regeneration of the oxidized FAD cofactor -> forming NADH (product 2)
- repeat
7
Q
citric acid cycle: overall cycle
A
- acetyl-CoA is condensed with oxaloacetic acid (alpha and beta carboxylic acid)
- claisen condensation via citric synthase -> methyl group of acetyl-CoA appears as a methylene group in citric acid (the product)
- citrate is dehydrated via aconitase to the enzyme bound product cis-aconitate (cis-aconitic acid)
- cis-aconitic acid is transferred to the next enzyme -> isocitrate dehydrogenase
- isocitrate- rehydrated product of cis-aconitic acid -> its hydroxyl group is move from the beta to the alpha -> alpha hydroxycarboxylic acid
- isocitrate dehydrogenase converts isocitrate to alpha-ketoglutarate -> loss of a CO2 (first loss of a carbon) -> decarboxylation
- alpha-ketoglutarate is oxidatively decarboxylated via alpha-ketoglutarate dehydrogenase complex -> forms succinyl-CoA (identical to pyruvate oxidative decarboxylation rxn)
- production of succinate from alpha-ketoglutarate passes through high energy mixed anhydrides and thioester
- succinyl-CoA synthetase (succinyl thiokinase) -> produces GTP by substrate level phosphorylation (from high energy mixed anhydrides and thioester)
- succinate is dehydrogenated on the inner membrane by succinate dehydrogenase (falvin dependent enzyme) -> produces fumarate
- fumarate (has a double bond) is hydrated to malate via fumarate
- malic acid is dehydrogenated by malate dehydrogenase (NAD dependent) -> forms oxaloacetate
- oxaloacetate is ready to participate in another round of cycle
8
Q
first half vs second half of krebs
A
- first half involves decarboxylation’s -> we lose 2 carbons
- second have is rehydration and dehydrations
- 2nd half is anabolic
- 2nd half is found in cancer cells
- the only decarboxylation in cancer cells during the krebs cycle is the alpha-ketoglutarate dehydrogenase step
9
Q
anaplerotic reactions
A
- nothing is made or destroyed -> intermediates are extracted though
- oxaloacetate can be extracted for gluconeogenesis
- glutamic acid can be converted to alpha-ketogluteric acid and supplement the species in the krebs cycle
- anaplerotic rxns deplete or enrich the krebs
10
Q
summary of sequence of events in the citric acid cycle
A
- 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 carboxylations to give 2 NADH
- step 5- substrate level phosphorylation to give GTP
- step 6 - dehydrogenation to have FADH2
- step 7- hydration
- step 8- dehydrogenation to give NADH
11
Q
citric acid cycle: step 1: C-C bond formation by condensation of acetyl-CoA and oxaloacetate
A
- condensation of oxaloacetate and acetyl-CoA via citrate synthase
- acetyl-CoA- thioester (high energy) -> instead of being used to make an ATP it goes into a condensation rxn and uses the energy to drive the rxn
- huge -ΔG -> -32.2 kJ/mol
- highly thermodynamically favorable/irreversible
- pushes the entire kreb cycle in one direction
- this is the only rxn with C-C bond formation
- uses acid/base catalysis
- carbonyl of oxaloacetate is a good electrophile
- methyl of acetyl CoA is a not a good nucleophile unless activated by deprotonation
- this is the rate limiting step
- activity largely depends on oxaloacetate
- product is citrate
- regulated by substrate availability and product inhibition -> bc oxaloacetate has other fates (gluconeogenesis or making malate which is more favored)
12
Q
induced fit in the citrate synthase (step 1 enzyme)
A
- conformational change occurs upon binding oxaloacetate
- ordered bisubstrate rxn
- avoid unnecessary hydrolysis of thioester in acetyl-CoA
- open conformation- binds oxaloacetate very well but free enzyme does not have a binding site for acetyl-CoA
- closed conformation- binding of OAA created binding for acetyl-CoA -> reactive carbanion is protected
- the idea that the enzyme cannot bind the second substrate until the first substrate is bound -> bisubstrate rxn with obligate binding order
- analog of acetyl-CoA is an uncompetitive inhibitor for oxaloacetate
- conformational change protects the product from premature hydrolysis
- enzyme hides substrates from water
- oxaloacetate binds to bottom of a site that creates an anchor for acetyl CoA to then finally enter the binding site
13
Q
citric acid cyce: step 2: isomerization by dehydration/rehydration
A
- hydroxyl group is moved from 2 position to 1 position
- citrate is converted to isocitrate via aconitase enzyme
- small ΔG
- not favored one way or another -> more unfavorable/reversible
- product concentration kept low to pull forward rxn
- isocitrate is active product -> L-isocitrate
- elimination of H2O from citrate gives cis double bond -> lyase
- citrate (tertiary alcohol) is a poor substrate for oxidation
- isocitrate (secondary alcohol) is a good substrate for oxidation
- addition of H2O to cis-aconitate is stereospecific
14
Q
iron sulfur center in aconitase
A
- water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center -> sensitive to oxidative stress
- only make L-isocitrate (optically active) from inactive citrate
- aconitase has iron sulfur center
- holds citrate in a specific conformation to define the dehydration of citrate to form cis-aconitate
- enzyme is asymmetric and can only bind in one orientation
- citrate is a prochiral molecules -> noramlly optically inactive but replacement of either -CH2COO- groups would make the central C chiral -> active
- aconitase is stereospecific
- augstin 3 point attachment model -> if the enzyme can bind to optically inactive substrate (citrate) at at least 3 points the product will be stereospecifically defined (L-isocitrate)
- possible to induce a center of chirality by binding a optically inactive substrate at at least 3 points
15
Q
citric acid cycle: step 3: oxidative decarboxylation by isocitrate dehydrogenase
A
- isocitrate is easily dehydrated via isocitrate dehydrogenase
- isocitrate dehydrogenase is a mitochondrial enzyme that makes use of NAD
- allosteric site for ATP -> inhibits mitochondrial enzyme
- ATP is the product of oxidative decarboxylation -> negative feedback
- oxalosuccinate is an enzyme bound intermediate -> beta keto acid -> spontaneously decarboxylate
- forms a 5 carbon dicarboxylic acid -> alpha-ketoglutarate (similar to alpha-keto acid pyruvate)
- forms NADH for ETC
- alpha-ketoglutarate is the product -> imported from glutamine or exported to glutamate -> regulation
- highly favorable and irreversible (but it is reversible in the cytosol with NADP -> cancer cells)
- some alpha-ketoglutarate is converted back to isocitrate
- cytosolic isocitrate dehydrogenase makes use of NADP -> this enzyme may run the rxn in the opposite direction in cancer cells -> produces isocitrate and then citrate -> citrate leaves into cytosol and carries out biosynthetic rxns
- catalyzes a rate limiting step -> speeds up the entire krebs in presence of ADP and Ca
16
Q
citric acid cycle: step 4: final oxidative decarboxylation by alpha-ketoglutarate dehydrogenase
A
- alpha-ketoglutarate (5 carbon dicarboxylic acid) converted to succinyl-CoA via alpha-ketoglutarate dehydrogenase complex
- this complex is very similar to pyruvate dehydrogenase complex -> all the same cofactors
- E1 and E2 are different to accommodate different sized substrates but E3 are identical
- favorable -> irreversible
- regulated by product inhibition
- oxidative decarboxylation of an alpha-keto acid -> TPP
- last oxidative decarboxylation
- net full oxidation of all carbons of glucose after 2 turns of the cycle
- carbons not directly from glucose bc carbons lost come from oxaloacetate, not acetate
- succinyl-COA is another higher energy thioester bond
17
Q
alpha-ketoacids making use of TPP
A
- alpha-ketoacids that are oxidatively decarboxylated used same mechanisms using TPP
- seen in pyruvate dehydrogenase complex -> acetyl-CoA
- citric acid cycle -> step 4 -> succinyl-CoA
- oxidation of isoleucine (leucine, valine) -> branched amino acids -> produces alpha-methylbutyryl-CoA
18
Q
alpha-ketoglutarate dehydrogenase complex
A
- three enzymes
- 5 cofactors
- E1- alpha-ketoglutarate dehydrogenase (TPP)
- E2- dihydrolipoyl transsuccinylase (lipoamide, CoA)
- E3- dihydrolipoyl dehydrogenase (NAD+, FAD)
- E3 from pyruvate dehydrogenase complex is identical to E3 from alpha-ketoglutarate dehydrogenase complex
- alpha-ketoglutarate reacts with TPP -> forms hydroxyethyl-TPP complex -> forms active succinyl intermediate
- active succinyl intermediate is transferred to lipoic acid to produce succinyl hydrolipoic acid
- succinyl hydrolipoic acid reacts with CoA -> forms succinyl-CoA (thioester) and dihydrolipoic acid
- E3=E3 of pyruvate dehydrogenase
- dihydrolipoic acid is reoxidized by disulfide -> forms an internal dithiol -> reoxidized by FAD -> which is reoxidized by NAD -> produces a NADH and reoxidized lipoic acid to repeat
- long lipollysyl arm transfers intermediates from one active site to another
- this rxn is abnormally low in people suffering from beriberi (deficiency of thiamine)
- 2 different coenzymes with sulfhydryl groups participate
19
Q
origin of C-atoms in CO2
A
- carbons from acetate are red
- all CO2 generated during the citric acid cycle is produced before succinyl-CoA is made
- in one turn of the citric acid cycle, neither of the red carbons is lost
- both CO2 molecules lost were present on the oxaloacetate used to begin the cycle
- citrate -> isocitrate -> alpha-ketoglutarate -> succinyl-CoA (6 carbons to 4 carbons)
- citrate is made from 4 carbon oxaloacetate and 2 carbons from acetyl-CoA
- the 2 carbons from acetyl-CoA are NOT the carbons released until we convert succinyl-CoA to succinate -> in this step the molecule loses it selectivity and structure
- carbons from acetyl-CoA are retained from conversion to citrate to succinyl-CoA
20
Q
citric acid cycle: step 5: generation of GTP through thioester
A
- substrate level phosphorylation by succinyl-CoA synthetase
- succinyl-CoA synthetase- going from succinate to succinyl-CoA (reverse)
- succinyl thiokinase- going from succinyl-CoA to free succinate (forward)
- conversion of succinyl-CoA to succinate
- free thiol of succinyl-CoA comes from CoA (thiol came from the enzyme itself from glyceraldehyde-3-dehydrogenase)
- it makes a phosphoenyzme and a mixed anhydride
- succinyl-CoA reacts with inorganic phosphate and the enzyme succinyl thiokinase -> forms an enzyme bound mixed anhydride -> uses this to make a phosphohistidiene on the enzyme -> phosphohistidyl enzyme
- succinate is released (symmetrical and inactive)
- phosphohistidiene is used to phosphorylate GDP to make GTP
- sulfhydryl is not contributed by the enzyme -> rather CoA
- similar to the way thioester was used by glyceraldehyde-3-phosphate dehydrogenase and diphosphoglycerate kinase -> made a mixed anhydride
- for the enzyme diphosphoglycerate kinase direct phosphorolysis succinyl-CoA by inorganic phosphate to make ATP -> but in this rxn the enzyme succinyl thiokinase puts the phosphate onto a histidine and then onto GTP (substrate level phosphorylation)
- acyl phosphate
21
Q
citric acid cycle: step 6: oxidation of an alkane to alkene by succinate dehydrogenase
A
- succinate converted to fumarate via succinate dehydrogenase
- succinate is optically inactive -> origin of the carbons become scrambled
- succinate dehydrogenase part of the mitochondrial inner membrane
- highly reversible (near equilibrium
- product concentration kept low to pull forward
- this enzyme is special bc its also an intermediate step in the electron transport chain -> acts as complex II in the ETC
- flavin dependent enzyme
- succinate reduced FAD - FADH2
- succinate itself becomes oxidized -> fumarate
- reduced of alkane to alkene requires FADH2
- reduction potential of carbon-hydrogen bond is too low for production of NADH
- FAD is covalently bound (unusual)
22
Q
citric acid cycle: step 7: hydration across a double bond
A
- addition of water to fumarate (fumaric acid) to L-malate (malic acid) via fumarase
- malate is chiral (optically active)
- fumarate is optically inactive -> converted to L-malate
- somewhat similar to 3 point model of aconitase
- slightly thermodynamically favorable -> highly reversible
- product concentration kept low to pull rxn forward
- fumarase is stereospecific
- addition of water to fumarate is always trans and forms L-malate
- OH- adds to fumarate -> then H+ adds to the carbanion
- cannot distinguish between inner carbons -> so either can gain the OH-
- if you give fumarase D-malate it will attempt to make maleate (maleaic acid) -> cis
23
Q
citric acid cycle: step 8: oxidation of alcohol to a ketone and regeneration of oxaloacetate by malate dehydrogenase
A
- L-malate is oxidized to oxaloacetate via L-malate dehydrogenase
- L-malate is capable of diffusing out of the mitochondrial membrane -> implications for gluconeogenesis
- unfavorable -> reversible
- citrate synthase has a low delta G -> favorable -> therefore this rxn is winning -> free oxaloacetate concentration is low in mitochondria -> pulls rxn forward
- final step of the krebs
- regenerates oxaloacetate for citrate synthase
- NAD reduced to NADH
- malate dehydrogenase helps overcome the impermeability of oxaloacetate to the mitochondrial membrane -> mitochondrial and cytosolic catalyze malate/oxaloacetate interconversion that are part of malate-oxaloacetate shuttle
24
Q
Net result of the citric acid cycle
A
acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O -> 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
- net oxidation of 2 carbons to CO2 -> equivalent of 2 carbons of acetyl-CoA -> but NOT the exact same carbons
- energy captured by electron transfer to NADH and FADH2
- generates 1 GTP -> can be converted to ATP
- completion of cycle
- source of GTP (ATP), CO2, and reducing equivalents (substrates for ETC -> ATP)
25
Q
CAC intermediates are amphibolic
A
- amphibolic- both catabolic and anabolic functions
- catabolic- oxidation of acetate to CO2
- anabolic- intermediates serve as precursors to other molecules
- depletion of intermediates for anabolic pathways creates a problem for catabolism -> slows CAC
- depletion come from biosynthesis in the cell
- beginning of an anabolic path for the cancer cell
- some intermediates can go in and out of mitochondria
- krebs cycle is amphibolic -> some rxns feed the cycle and some deplete the cycle
- glutamine is used by the mitochondrion of many cancer cells and converted to glutamate and then to alpha-ketoglutarate (alpha-ketoglutarate can leave the mitochondria)
- cancer cells rely heavily on glutamine to run the second half of the krebs cycle
- involving multiple forms of isocitrate dehydrogenase we can run the first half of the krebs cycle backwards up to citrate
- citrate can leave the mitochondria -> citrate is a powerful inhibitor of glycolysis -> especially a potent inhibitor for glyceraldehyde-3-phophsate dehydrogenase
- malic acid can go in and out of the mitochondria
- oxaloacetate leaves the mitochondria by being converted to malic acid to be used but is also used inside the mitochondria for other purposes
- oxaloacetate is the keto acid that is formed from aspartic acid
- alanine (amino acid) is formed from pyruvate (keto acid)
- many amino acids can be converted to glucose -> glucogenic amino acids
- some amino acids can only be converted to kreb cycle intermediates -> ketogenic
26
Q
cataplerotic reactions
A
- empty the citric acid cycle
- depletion
- use of citrate in metabolism
- aspartyl aminotransferase converts oxaloacetate + alanine aspartate + pyruvate -> enzymes leak out when liver is damaged
- glutamate dehydrogenase converts alpha-ketoglutarate +NADH + H+ + NH4 glutamate + NAD+ + H2O
27
Q
anaplerotic reactions
A
- fill the citric acid cycle
- doesnt provide the substrate acetyl-CoA -> provides intermediates
- pyruvate carboxylase is rxn that creates oxaloacetate (kreb cycle intermediate)
- amino acid breakdown
- odd-chain fatty acid breakdown
- pyruvate + CO2 + ATP + H2O -> oxaloacetate + ADP + Pi
- acetyl-CoA activates pyruvate carboxylase, makes more oxaloacetate and then permits pyruvate to be used as a source of both substrate and intermediate for krebs cycle
- intermediates in the CAC can be used in biosynthetic pathways (removed from cycle)
- must replenish intermediates in order for cycle and central metabolic path to continue
- 4-carbon intermediates are formed by carboxylation of 2-carbon precursors
- involves fixation of CO2
28
Q
pyruvate carboxylase: anaplerotic reaction
A
- pyruvate + CO2 + ATP + H2O -> oxaloacetate + ADP + Pi
- liver and kidney
- replenish krebs
29
Q
PEP carboxykinase: anaplerotic rxn
A
- phosphoenolpyruvate + CO2 + GDP -> oxaloacetate + GTP
- heart and skeletal muscle
- replenish krebs
30
Q
PEP carboxylase: anaplerotic rxn
A
- phosphoenolpyruvate + CO2 -> oxaloacetate + Pi
- higher plants, yeast, and bacteria
- replenish krebs
31
Q
malic enzyme: anaplerotic rxn
A
- pyruvate + CO2 + NAD(P)H -> malate + NAD(P)
- widely distributed in eukaryotes and bacteria
- both in mitochondria and cytosol
- involves biotin
- 3-carbon -> 4- carbon
- replenish krebs
32
Q
regulation of citric acid cycle
A
- regulated at highly thermodynamically favorable and irreversible steps -> PDH, citrate synthase, IDH, and KDH
- activated by substrate availability
- inhibited by product accumulation
- overall products of krebs (NADH and ATP) affect all regulated enzymes
- inhibitors- NADH and ATP
- activators- NAD+, ADP and AMP
- pyruvate dehydrogenase to make acetyl-CoA -> can be phosphorylated at E1 -> ATP inhibits -> inhibits krebs
- acetyl-CoA is a potent feedback inhibitor of pyruvate dehydrogenase as well
- fatty acids dont directly inhibit pyruvate dehydrogenase but they are metabolized to acetyl-CoA -> inhibits
- citrate synthase is feedback inhibited by citrate and also inhibited by succinyl-CoA
- isocitrate dehydrogenase is very sensitive and inhibited by ATP -> stimulated by ADP -> therefore the reducing equivalents will be convert ADP to ATP -> inhibits
33
Q
regulation of pyruvate dehydrogenase
A
- regulated by reversible phosphorylation of E1: phosphorylation -> inactive and dephosphorylation -> active
- PDH kinase and PDH phosphatase (a form of phosphoprotein phosphatase) are part of mammalian PDH complex
- kinase is activated by ATP
- high ATP -> phosphorylated PDH -> less acetyl-CoA
- low ATP -> kinase is less active and phosphatase removes phosphate from PDH -> more acetyl-CoA
- regulation of PDH is somewhat similar to regulation of glycogen synthase/glycogen phosphorylase system by protein kinase cascade and phosphoprotein phosphatase
34
Q
regulation of citrate synthase
A
- -citrate synthase is feedback inhibited by citrate and also inhibited by succinyl-CoA
- alpha-ketoglutarate is an important branch point for amino acid metabolism
- succinyl-CoA communicates flow at this branch point to the start of cycle
35
Q
regulation of isocitrate dehydrogenase
A
- controls citrate levels
- very sensitive and inhibited by ATP -> stimulated by ADP -> therefore the reducing equivalents will be convert ADP to ATP -> inhibits
- aconitase is reversible
- inhibition of IDH leads to accumulation of isocitrate and reverses aconitase
- accumulated citrate leaves mitochondria and inhibits phosphofructokinase in glycolysis
36
Q
krebs, glycolysis, and cancer
A
- cancer cells accumulate succinate (from succinyl thiokinase) and fumarate (from fumarase) as a result of loss of function of fumarase and succinic dehydrogenase (mutated)
- excess succinate chemically modifies enzymes involved in DNA and histone methylation -> succinylation
- cancer cells have NADP-dependent isocitrate dehydrogenase isozymes (in cytosol) with elevated activity leading to accumulation of alpha-hydroxyglutarate (2-HG)
- inhibition of alpha-ketoglutarate-dependent oxygenases by 2-HG results in extensive epigenetic modification to DNA in cancer
- cancer cells accumulate glutamine -> convert it to alpha-ketoglutarate, malate, pyruvate, and finally lactate, which is secreted in high amount by cancer cells
37
Q
isocitrate dehydrogenase in cancer cells
A
- cytosolic
- take alpha-ketoglutarate and reduce it to alpha-hydroxyglutarate
- NADP-dependent rxn
38
Q
alpha-hydroxyglutarate
A
- produced from isocitrate dehydrogenase in the cytosol for cancer cells
- methylates DNA and RNA
- methylates histones
- alter the functional properties of cancer cells associated with loss of contact inhibition
- alters the extracellular matrix in cancer cells
39
Q
cancer cells take up glucose and glutamine
A
- cancer cells can do things with malic acid that is formed from the glutamine they take up -> export it to make pyruvate and lactate
- accounts for high levels of high lactic acid production in cancer cells
40
Q
warburg effect
A
- even though cancer cells may have a lot of O2 available -> they still tended to convert (ferment) glucose into lactate
- the glucose that is being metabolized through the glycolytic pathway up to a certain point -> pyruvate kinase
- lactate is not a product of synthesis of pyruvate by the cancer cells -> rather the lactate is a product of the malate that is carrying second part of krebs forward
- the reason the pyruvate is converted to lactate cant be derived all from glucose is that -> in the step that converts phosphoenolpyruvate to pyruvate -> many cancer cells have a M2 variant of pyruvate kinase -> sluggish
- PEP accumulates or relys of glycolysis and all the glycolytic intermediates accumulate
- PEP can be used to make the phosphoglycerate mutase that converts 3-phosphoglyerate to 2-phosphosphoglycerate
- PEP continuously converts 3PG to 2PG by donating a phosphate group to the histidine of phosphoglycerate mutase (rather than ATP)
41
Q
M2 isoform
A
- people tried to make activators for M2 isoform of pyruvate kinase to proceed through glycolysis and make pyruvate for krebs
- cancer arnt interesting in feeding their mitochondria with glycolytic intermediates -> they want to use glycolysis to make more intermediates for biosynthetic rxns
42
Q
malaria
A
- parasite (plasmodium falciparum) can be eliminated by red cells that have undergone structural changes as a consequence of -> ex. hemoglobin S disease of glucose-6-phosphate dehydrogenase deficiency
- plasmodium falciparum does not carryout synthesis of acetyl-CoA for krebs cycle efficiently uses pyruvate and pyruvate dehydrogenase (unlike cancer cells) -> it feeds its krebs cycle with alpha-ketoglutarate (like cancer cells) from glutamic acid and glutamine
- uses the acetly-CoA to make amino sugars
- one it has made alpha-ketoglutarate and put it in the krebs -> it will secrete malate (like cancer cells)
- runs the second half of the krebs cycle in the forward direction up to the point of malate -> exports that malate
- uses the other isoform of isocitrate dehydrogenase to run the first half of krebs backward
- second half of krebs is a dehydrogenation pathway -> makes reduced products/equivalents -> if we run the 1st half of krebs backward we need to oxidize the ETC intermediates
- NO net gain of oxidation or reduction of ETC intermediates in malarial parasites -> no significant oxidation phosphorylation (ATP)
- this is ok bc it doesnt really have mitochondria it does krebs cycle in cytosol (makes minimal ATP in mitochondrion)
- mimics the metabolic events in a mammalian cancer cells -> conversion of pyruvate to lactate and running of the krebs by feeding it with alpha-ketoglutarate
43
Q
ETC overview
A
- intermediates have been reduced
- intermediates capture electrons for redox rxns
- generates a proton motor force -> generated by movement of electrons along the ETC
- equal distribution of proton on either side of the inner membrane is responsible for synthesis of ATP
44
Q
energy from reduced fuels are used to synthesize ATP in animals
A
- carbohydrates, lipids, and amino acids are the main reduced fuels for the cell -> get oxidized
- electrons from reduced fuels are transferred to reduced cofactors NADH or FADH2 -> drive ETC
- generate a proton gradient -> used to make ATP
- in oxidative phosphorylation, energy from NADH and FADH2 is used to make ATP
45
Q
chemiosmotic theory
A
- ADP + Pi -> ATP is a highly thermodynamically unfavorable
- phosphorylation of ADP is not a result of a direct rxn between ADP and some high energy phosphate carrier
- energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient -> driving force
- distributed unequally
- energy released by ETC is used to transport protons against the electrochemical gradient
- captures a series of oxidations of the reduced cofactors -> provides energy to establish unequal distribution of proton across the inner membrane
46
Q
chemiosmotic energy coupling requires membranes
A
- proton gradient needed for ATP synthesis can be stably establish across inner membrane that is impermeable to ions
- membrane must contain proteins that couple the downhill flow of electron in the electron transfer chain with the uphill flow of proton across the membrane
- membrane must contain a protein that couples the subsequent downhill of protons to the phosphorylation of ADP
- intermembrane transporters have the ability to move electrons from their reduced state to oxidized state
- vectorial protons
- movement of vectorial protons -> membrane bhor effect
- proteins of ETC that bind or release protons (chemical/scalar protons) themselves through oxidation and reduction -> stochiometric
- proteins undergo conformational changes -> allows proteins to move protons from the matrix through the intermembrane to the intermembrane space
- vectorial and scalar protons are generated as a result of the movement of the electron down the ETC -> proton gradient is used to drive the ATP synthase (dissipates gradient)
- ATP synthase is induced to undergo conformational changes that causes phosphorylation of ADP -> ATP
47
Q
ATP synthase
A
- proton gradient is used to drive the ATP synthase
- dissipates proton gradient
- induced to undergo conformational changes in its F0 subunit -> causes phosphorylation of ADP -> ATP
48
Q
structure of mitochondrion
A
- double membrane leads to 4 distinct compartments
- outer membrane- relatively porous membrane that allows passage of metabolites
- intermembrane space (IMS)- similar environment to cytosol and has a higher proton concentration (lower pH)
- inner membrane:
- relatively impermeable (even to protons), with proton gradient across it
- location of ETC complexes (4)
- convolution called cristae serve to increase the SA
- matrix: location of the krebs and parts of lipid amino acid metabolism (oxidation) -> lower proton concentration (higher pH)
49
Q
FMN and FAD: electron funnels
A
- FMN and FAD can be covalently bound to proteins and act to funnel and distribute electrons to ETC
- flavins
- accept 2 electrons from carrier (NADH and NADPH) that are unstable donated single electrons
- can donate 1 electron at a time, using a semiquinone-like free radical intermediate to acceptors that can only accept single electrons -> sometimes good sometime risky
50
Q
cytochromes
A
- all cytochromes are one electron carriers
- either in ferric or ferrous state
- contains hemes
- iron coordination porphoryin ring derivatives
- feroprotoporphyrin 9 -> in hemoglobin -> this can be oxidized or reduced in the b-type cytochrome
- a. b, or c differ by ring additions
- heme A has a long hydrophobic chain that anchors it to the membrane of the last component of the ETC (complex 4)
- heme C is associated with the c-type cytochromes -> completely soluble and not bound to any membrane
- although c-type cytochrome has a binding site on the inner membrane
- cytochrome C1 (a different type) -> we find in complex 3
- cannot diffuse through (within) membranes but it can bind
- mobile carrier bc it moves from complex to complex
51
Q
iron sulfur clusters
A
- one electron carriers
- iron is coordinated to cysteines in the protein
- there are multiple iron sulfur centers or clusters each with a distinct arrangement of sulfur atoms coordinated to the iron
- iron and sulfur (given from cysteine) are holding the clusters together
- moving electrons to the other one electron carriers like cytochromes
- participate in some rxns in which the valency is not strict -> mixture of ferous and feric iron -> imperfect stoichiometry’s of electron movement (in general one electron carriers though)
- many iron-sulfur centers contain multiple irons -> means that the electrons that are carried can move within the centers -> not just redox intermediates -> they are truly carriers that can move electrons through proteins
52
Q
coenzyme Q or ubiquinone
A
- ubiquinone (CoQ) is a lipid soluble conjugated dicarbonyl compound that readily accepts electrons
- 2 electron donor
- hydrophobic -> never leaves the internal bilayer of membranes -> floats within
- mobile electron carrier -> goes to complex to complex
- upon accepting 2 electrons, it picks up 2 protons to give an alcohol -> ubiquinol (CoQH2)
- mechanism acquires protons selectively from one or the other side of the inner membrane
- reoxidation of ubiquinol to ubiquinone releases the protons selectively to another side of the inner membrane
- CoQ and CoQH2 can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side
- CoQ pool is larger than the number of complexes in the ETC
- Coenzyme Q is a mobile electron carrier transporting electrons from complexes 1 and 2 to complex 3
- can exist in quinone form (fully oxidized), semi-quinone form (reduced by 1 electron), or quinol (hydroquinone) form (fully reduced by 2 electrons)
- there is always a ubiquinone available to participate in a redox rxn
53
Q
complex 1 of ETC
A
- NADH dehydrogenase
- contains a flavin
- contains a lot of iron sulfur clusters
- no cytochromes
54
Q
complex 2 of ETC
A
- succinate dehydrogenase
- from krebs cycle (only part of krebs that is membrane bound)
- iron sulfur clusters
- FAD
55
Q
complex 3 of ETC
A
- ubiquinone: cyto
- b and c type hemes/cytochromes
- iron sulfur clusters -> rieske center -> one of the irons is coordinated by 2 histidine’s
- contains histidine and cysteine
- cytochrome c participates
56
Q
complex 4 of ETC
A
- cytochrome oxidase
- contains 2 different kinds of a-type cytochromes
- 2 copper atoms
57
Q
NADH: Ubiquinone Oxidoreductase (Complex 1)
A
- one of the largest macromolecular assemblies in the mammalian cell
- over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes
- NADH binding site facing the matrix side
- noncovalently bound flavin mononucleotide (FMN) accepts 2 electrons from NADH
- several iron sulfur centers pass 1e- at a time toward the ubiquinone binding site (from NADH) -> forms matrix arm
- matrix arm does electron transport
- when the electron reaches the ubiquinone binding site (bound loose) it passes to the ubiquinone one at a time -> makes a semiquinone -> then the ubiquinol (QH2)
- QH2 picks up 2 protons
- QH2 stays in the intermembrane bc its so hydrophobic
- protons are transported by proton wires (membrane bohr effect)
- transmembrane proteins- responsible of movement of vectorial protons from matrix (N) to intermembrane space (P)
- bind protons on the matrix side and release them on intermembrane space -> membrane like bhor effect
- the protons that are moved are bound and released by ionizable amino acids (protonated and deprotonated) within the component of the membrane arm to get a net transfer of a proton from one side of a membrane to another
- membrane arm move about 4 protons per 2 electrons (or 1 NADH) moved through matrix arm
- NADH + Q + 5H+ = NAD+ + QH2 + 4H+
- transmembrane proteins undergo conformational changes -> drives proton pumping mechanism
- conformational changes alter the pK values of ionizable side chains so that protons are taken up or released as electrons are transferred
58
Q
energy produced by complex 1
A
- there is enough energy to make an ATP from the reduction of CoQ by NADH
- ADP cant bind to any of the species therefore it is not made
- there is more than enough energy captured in the movement of the protons
59
Q
inhibition of complex 1
A
- inhibited by rotenone or amytal
- rotenone- rat poison
- bind to complex 1
60
Q
succinate dehydrogenase: complex 2
A
- succinate binding site in the matrix
- succinate is oxidized to fumarate -> 2e- picked up by FAD
- succinate dehydrogenase is covalently bound to FAD
- FAD accepts 2e- from succinate and donates 1e- at a time
- electrons are passed one at a time via iron sulfur centers (and 1 cytochrome b) to ubiquinone which becomes reduced to QH2
- QH2 goes into the inner membrane -> floating
- does not transport protons (no membrane arm)
- free energy from succinate to CoQ is insufficient to drive ATP synthesis -> still important bc it allows relatively high potential e- to enter ETC by bypassing complex 1
- can move 2e-
- adds to the pool of reduced ubiquinone
- succinate dehydrogenase is a single enzyme with dual roles
- coverts/oxidizes succinate to fumarate in the citric acid cycle (reducing FAD to FADH2)
- captures and donates electrons in the ETC
- heme b (nothing to do with e- transport)- scavenges free radicals that might have formed as a result of e- movement such as semi-quinone in FAD
61
Q
complex 1 and 2
A
- do not operate in series
- they happen side by side
- not one after the other
- both accomplish the same result: transfer of electrons to CoQ from reduced substrate (NADH or succinate)
- CoQ diffuses in the lipid bilayer among the respiratory complexes and serves as a collection point for electrons
- CoQ collects electrons from the FADH2 produced by the glycerophosphate shuttle
62
Q
intermembrane space
A
-higher concentration of protons when electrons are allowed to pass through the entire ETC
63
Q
matrix
A
-lower concentration of protons when electrons are allowed to pass through the entire ETC
64
Q
double duty of ETC
A
- moves electrons down electrochemical gradient
- moves protons
65
Q
ubiquinone: cytochrome c oxidoreductase: Complex 3
A
- uses 2e- from QH2 to reduce 2 molecules of cytochrome c
- cytochrome c is not apart of any mitochondrial membrane (floats in intermembrane space)
- contains iron sulfur clusters, cytochrome b isoforms, and cytochrome c
- clearance of electrons from the reduced quinones via the Q-cycle results in translocation of 4 additional protons to the intermembrane space
- cytochrome b(Low) -> contains bL type heme
- cytochrome b(High) -> contains bH type heme
- two binding sites for CoQ -> Qp/o and Qn/i
- Qp/o- pointing towards intermembrane space/positive space (protons here)
- Qn/i- pointing towards the matrix/
- rieske center is here- alternatly donate electrons to cytochrome bL or bH or cytochrome c1
- cytochrome c1 (has heme c1)- has a basket at the top that points into the intermembrane space and binds cytochrome C
66
Q
rieske center
A
- iron is coordinated to 2 cysteines and histidine’s
- functions to alternating donate electrons to cytochrome bL or bH or cytochrome c1
67
Q
the Q cycle: complex 3
A
- 4 protons are transported across the membrane per 2 electrons that reach cytochrome c
- 2 of the 4 protons come from QH2 (from complex 1 or 2) the other 2 come from the matrix
- Q cycle provides good model that explains how 2 additional protons are picked up from the matrix
- depleting the matrix of protons
- 2 molecules of QH2 become oxidized -> releases protons into the intermembrane space
- one molecule becomes re-reduced -> net transfer of 4 protons per reduced CoQ
- protons are NOT moved by conformational changes in the proteins here, -> oxidation and reduction of CoQ moves them -> these are chemical or scalar protons
- 2 molecules of cytochrome C are reduced to the ferrous form
68
Q
Q cycle: complex 3: cycle 1
A
- reduced QH2 from complex 1 or 2
- reaction involving rieske center separates the electrons in QH2 -> converts to fully oxidized CoQ -> diffuses back into bilyaer
- the 2 protons are unidirectionally diffused into the intermembrane space
- protons are moved across inner membrane by a lipid soluble carrier (QH2) that occupies 2 sites
- one of the electrons from QH2 is passed to cytochrome C1 and then to cytochrome C
- the other electron is passed to cytochrome bL and then to cytochrome bH -> finally transferred to oxidized CoQ
- oxidized CoQ is converted to a free radical (semiquinone)
- QH2 + Q + cyt c (oxidized) -> Q + Q- + 2Hp + cyt c (reduced)
69
Q
Q cycle: complex 3: cycle 2
A
- another fully reduced QH2 enters and is split
- 2 protons from the QH2 are unidirectionally deposited into the IMS
- 1 electron is passed to reduced cytochrome c1 and then cytochrome C
- the other electron is passed to cytochrome bL and cytochrome bH -> then the electron reacts with the free radical (semiquinone) that was generated in the first cycle of Q cycle
- electron reduces the semiquinone and picks up 2 protons unidirectionally from the matrix
70
Q
inhibitors of complex 3
A
- antimycin A- binds to the Qi/n site (site that is closest to the mitochondrial matrix)
- stigmatellin- binds to the Qo/p site (site that binds CoQ that is very close to the intermembrane space)
- antimycin blocks electron transfer between cytochrome b and c1
71
Q
cytochrome C
A
- second mobile electron carrier
- one electron acceptor
- never enters the membrane
- soluble heme-containing protein in the intermembrane space
- heme iron can be either ferrous (oxidized) or ferric (reduced)
- cytochrome c carries a single electron from the cytochrome bc1 complex in complex 3 to cytochrome oxidase (complex 4)
- can accept electrons directly from an iron containing heme on one complex to a copper on another complex without any intermediates or cofactors
72
Q
cytochrome c absorbs visible light
A
- intense Soret band near 400nm absorbs blue light and gives cytochrome c an intense red color
- cytochromes are named by the position of their longest-wavelength (alpha) peak
73
Q
cytochrome oxidase: complex 4
A
- mammalian cytochrome oxidase is a membrane protein with 13 subunits
- contains 2 heme groups: a and a3
- contains copper ions:
- CuA: 2 ions that accept electrons from cytochrome C
- CuB: bonded to heme a3, forming a binuclear center that transfers 4 electrons to oxygen (tyrosine involved in binding)
- copper ions are held in place by histidine’s and a cysteine -> a tyrosine is involved too for CuB
- one the protein subunits it capable of moving protons through a membrane type bhor effect -> vectorial/ pumped protons
- there are also scalar/chemical protons too -> these arnt deposited into the IMS, rather they are depleted from the matrix -> used to reduced oxygen to water
- hydrogens of the water are obtained specifically from the matrix by scalar protons
74
Q
cytochrome oxidase passes electrons to O2: complex 4
A
- 4 electrons are used to reduce 1 oxygen molecule into 2 water molecules
- 4 protons are picked up from the matrix in this process -> chemical/scalar protons
- 4 additional protons are passed from the matrix to the intermembrane space via membrane bohr effect (conformational changes) -> vectorial/pumped protons
75
Q
electron flow through complex 4
A
- 4 electrons from cytochrome C and passes them to CuA
- CuA gives the electrons to heme A
- electrons then move from heme A to a complex including heme A3 and CuB
76
Q
complex 4: formation of water
A
- O2 bridges CuB to heme a3 -> splitting of this leads to H2O
- O2 binds to site with heme iron, non-heme copper, and tyrosine that can form a free radical
- electrons move from CuA to heme a to the heme a3 and CuB complex
- iron and copper conspire to split O2 into two O units that can each be protonated by scalar protons
- coppers go from Cu(1) (cuprous) to Cu(2) (cupric)
- iron in heme a3 exists in 3 valences -> ferrous, ferric, and ferril (Fe+4)
- all of the different valences of iron and copper must be achieved to split O2
- complex 4 carries a series of 1e- rxns involving changes in valency of Cu and Fe atoms
77
Q
alternate donor of electron to complex 4
A
- rather than just cytochrome C there is tetramethyl-p-phenylenediamine (TMPD)
- can donate electrons to complex 4 directly
- we can bypass complex 1, 2, and 3 with TMPD
- electron pair is initially derived from ascorbic acid (reduces 2 electrons at a time) which reduced TMPD
- cytochromes involved in complex 3 and 4 are 1 electron redox carriers (problem bc ascorbic is 2e-)
- TMPD is a mediator -> can carrier 2e- or 1e- at a time
- combination of ascorbate and TMPD can reduced cytochrome oxidase without the aid of complex 1, 2, and 3
78
Q
summary of ETC transport
A
- complex 1 -> comple 4
- 1NADH + 11H+ + 1/2O2 -> NAD+ + 10H+ + H2O
- complex 2 -> complex 4
- FADH2 + 6H+ + 1/2O2 -> FAD + 6H+ + H2O
- difference in number of protons transported relfects differences in ATP synthesized
- in reality these are rarely truly integral bc the vectorial protons are not participating directly in chemical rxns
- more protons are moved when complex 1 is the initial source of electrons than when complex 2 is used
79
Q
multiple complexes associate together to forma respirasome
A
- all the complexes together in a respirasome
- ensures great efficiency for electron movement from one complex to another
- not covalently linked -> they are aggerated in a multicomplex structure
80
Q
mitochondria
A
- couples electron transport using the various intermediates of the ETC
- also moves the protons
- protons are the driving force for ATP synthesis in the mitochondria
- ETC- oxidative part is carried out and reduction of water to O2
- ATP synthase carries out the phosphorylation part
81
Q
inhibitors of the ETC
A
- rotenone and amytal inhibits complex 1 -> block it at the conversion of NAD to NADH and the oxidation of NADH reducing CoQ
- no inhibitors of complex 2
- antimycin A and stigmatellin inhibits complex 3 -> block the reoxidation of reduced CoQ and the reduction of cytochrome C
- carbon monoxide (CO) and cyanide (CN-) (like hemoglobin) inhibited complex 4 moves protons using a membrane bohr effect and depletes the matrix of protons to complete the reduction of water to O2
- cyanide binds to ferric state iron -> irreversible
- a-type cytochrome heme is sensitive to cyanide
82
Q
proton motive force
A
- protons in ETC create electrochemical proton gradient by:
- actively transporting protons across the membrane (complex 1 and 4 -> vectorial protons)
- chemically removing protons from the matrix (reduction of CoQ in complex 3 and reduction of O2 to water in complex 4 -> scalar or chemical protons)
- releasing protons into the intermembrane space (oxidation of QH2 in complex 3 -> scalar or chemical protons)
- intermembrane space becomes positive with protons
- matrix is depleted and negative relatively (either into the intermembrane or by O2)
- proton concentration and electrical charge change -> chemical and electrical potential
- binding of protons from matrix to inner membrane proteins coupled to release of protons from other inner membrane proteins or other domains of membrane spanning protein into the intermembrane space
- rxn of protons in the matrix with ETC components without subsequent release into IMS
83
Q
chemiosmotic model for ATP synthesis
A
- electrical potential drops are balanced to the fact that protons are moving from the matrix to the intermembrane
- electropotential- charge separation
- chemical potential- proton concentration difference
- electron transport sets up a proton motive force
- energy of proton motive force drives synthesis of ATP
84
Q
ATP synthase (complex 5)
A
- 2 major components: F1 and F0
- F1- multiprotein component that sticks into the matrix -> can be split off the F0 component
- F1 is soluble in the matrix
- F1 individually catalyzes the hydrolysis (phosphorylation) of ADP and inorganic phosphate to ATP
- F0- anchored (integral) within the inner membrane
- multisubunit bridge that connects F0 and F1
- F0 transports protons from IMS to matrix dissipating the proton gradient -> energy is transferred to F1 to catalyze phosphorylation of ADP
- stalk- attached to the F0 and makes it way through the interior of F1
- driving force is the passage of protons through proton channel in F0
85
Q
relationship of ETC and ATP synthesis
A
- ATP synthesis via oxidative phosphorylation requires electron transport
- electron transport DOES NOT require ATP
- need for ADP and inorganic phosphate for the coupled preparation of mitochondria to take up O2
- when we add succinate substrate to ETC we see there is a large uptake of O2 and ATP is synthesized
- if we add succinate without ADP (substrate for ATP synthase) we see some electron transport and minimal ATP synthesis
- once ADP is added O2 is consumed and ATP is made
- when we add an inhibitor of the ATP synthase (oligomycin) -> no ATP is made and little O2 is taken up -> inhibits the entire ETC too!
- if we add dinitrophenol (DNP) which allows electrons to flow back in the matrix bypassing ATP synthase -> no ATP synthesis but still O2 is taken up -> inhibits ATP synthase but not the ETC
86
Q
uncouplers
A
- lipophilic weak acids
- pass through the membrane in either a charged or uncharged state
- when there are more protons in the IMS than matrix -> protons bind to uncouplers and are moved back into the matrix down the concentration gradient (no channels)
- allow protons to move back form the intermembrane to the matrix bypassing the ATP synthase
- collapses a proton gradient -> no driving force for ATP synthase (not essential for ETC)
- collapsing gradient prevents ATP synthesis but ETC is still carried out
- no ATP synthesis but still O2 is taken up
- inhibits ATP synthase but not the ETC
- DNP- 2,4-dinitrophenol (shoe polish)
- FCCP
- CCCP
87
Q
energy of the ETC
A
- there is more than enough energy that is associated with moving electrons from NADH all the way to reduction of H2O to O2
- enough to make 3 ATP worth but no ATP is made directly by the ETC
- big delta G
88
Q
structure of ATP synthase complex 5
A
- base is made up of 10-12 C subunits -> F0 that is anchored in the membrane
- F0 takes protons that are accumulated in the IMS into the matrix
- F1 has alpha and beta subunits
- gamma stalk connects F1 and F0 -> anchored in F0 and travels through the interior of F1 (not covalently attached)
- oligomycin sensitivity conferring protein (OSCP)- the top of the bridge
- conformational changes in the bridge are felt by the OSCP -> affects the alpha beta pairs of F1
- OSCP can cause a slowdown and stoppage of ETC in a well coupled mitochondrion if oligomycin is allowed to bind to complex V -> but ETC can resume if CCCP is then added to mitochondrion
89
Q
rotation of the gamma stalk
A
- attached to the C subunits of F0 and passes through the interior of F1 alpha beta pairs
- c subunits can undergo rotation that is structurally coupled to rotation of gamma subunit
- rotates
- first event in the synthesis of ATP is for ADP and inorganic phosphate to bind to one of the alpha beta pairs in the loose (L) conformation
- as the gamma stalk rotates alpha beta subunit pair goes through conformational change to the tight (T) site induced by the rotation of the gamma stalk -> brings the ADP and inorganic phosphate up against each other -> orbital steering (orbits line up)
- alpha-beta pairs in F1 DO NOT rotate
- ADP and inorganic phosphate react to form ATP
- additionally, in this step ATP’s previously made in the tight (T) conformation are now in the open (O) site -> escape of ATP
- gamma stalk rotates again and the 3rd alpha beta pair participates in binding ADP and inorganic phosphate in loose -> orbital streeing and rxn in the tight -> and release of ATP in the open
- repeat
- all of the ATP appears to come from the conformational changes in the alpha beta pairs induced by rotation of the gamma stalk
90
Q
isolated ATP synthase experiment
A
- F1 is anchored via the bridging peptide sequence -> His tag to an immobilized surface
- protons are supplied to the C subunits
- F-actin tail is attached to the C subunits
- F-actin tail rotates as the C subunits are offered protons
- the system can be made to rotate backwards by supplying ATP -> ATPase activity of the alpha beta pairs will rotates the entire system backwards
- proton gradient rotates forward
- if the complex is isolated so there is no gradient -> ATP can force it to rotate backward
91
Q
transport of ADP and Pi into matrix: substrates
A
- ATP synthase is driven by proton but it has substrates
- substrates are ADP and inorganic phosphate
- IMS and matrix are relatively impermeable
- ADP makes it way into the matrix from the IMS via an ATP ADP channel (antiporter)
- once ATP is made by F1 it escapes the matrix into the IMS and ADP is exchanged into the matrix
- inorganic phosphate is transported via symporter (moves inorganic phosphate and protons to maintain a charge balance)
92
Q
inhibitors of the ATP ADP antiporter
A
- atractyloside
- bongkrekic acid
- if you cant get substrates (ADP) into the matrix the synthase wont work
- you need both substrates and proton gradient
93
Q
positively charged residues
A
- responsible for moving the protons from the IMS through the C subunits back into the matrix
- ex. arg
94
Q
P/O ratios
A
- how many ATPs are made per O2 consumed by the ETC
- enough energy going from NADH all the way to reduction of H2O to O2 to make 3 ATP
- you can short circuit the chain by providing succinate instead of NADH -> fewer protons are moved from the matrix to the IMS -> then only 2 ATPs are made per O2 consumed
- if we use TMPD which bypassing almost the entire chain except for cytochrome oxidase -> just enough protons are moved by cytochrome oxidase to make 1 ATP (tested by using antimycin A and stigmatellen which inhibit complex 3)
- P/O ratio is consistent which the extent of rotation of the C subunits by the protons passing through the c subunit channels
- about 10 protons need to be passed through that channel for the complete rotation of the c-ring and gamma subunit -> makes 3 ATPs
- if we use succinate there will only be 2/3rds of a rotation -> 2 ATP made
- if we use ascorbate and TMPD there will only be 1/3rd of a rotation -> 1 ATP
95
Q
inhibiting the ETC by inhibiting the ATP synthase in a well coupled mitochondria
A
- if you were to inhibit ATP synthesis with oligomycin in a well coupled mitochondrial preparation -> you would no longer be able to allow protons to rotate the c subunits as they try to pass through the ATP synthase
- oligomycin inhibits by attaching to delta subunits of F1
- rotation of the c subunits is stopped my oligomycin
- no protons could pass through c subunits -> proton gradient being made from the ETC will grow bigger and bigger
- proton gradient is so large from high concentration of the proton in the IMS -> that the entire ETC would stop
- possible to stop electron transport in a well coupled mitochondria by inhibiting the ATP synthase
- if we add DNP, DCCP, CCCP -> destroys the gradient and now the ETC will proceed
96
Q
uncouplers: weight loss
A
- shoe polish- DNP
- CCCP
- FCCP
- used as weight loss strat
- oxygen is consumed (burn metabolites) in the ETC but no ATP is not made
- causes toxic problems
97
Q
natural uncouplers in the body
A
- UCP’s
- UCP2 and UCP3
- natural proteins
- partially in a limited way collapses the proton gradient
- work the same as CCCP and DNP
98
Q
ATP/ADP and ATP/Pi ratios
A
- in a well coupled mitochondrial preparation that is well supplied with substrates -> the ATP/ADP ratio and ATP/Pi ratio will be high bc ATP is being made
- the synthesis of ATP by a well coupled mitochondrial system is dependent on the source of ETC substrates
- NADH/NAD ratio affects this
- when NADH is high -> driving force to make ATP
- when NAD is high -> less ATP is made
- NADH/NAD ratio controls many steps in carbohydrate metabolism and krebs
- when NADH/NAD ratio is high -> citrate synthase, isocitrate dehydrogenase, and succinyl thiokinase are activated -> drives krebs to generate reducing equivalents which feed into ETC -> drives ATP synthesis
- when NADH/NAD ratio is low there are no proton to form the gradient to make ATP
- NADH/NAD ratio has reciprocal affects to ATP/ADP and ATP/Pi ratios
99
Q
IF1
A
- regulates ATP synthase
- at lower pHs IF1 inhibits ATPase activity of F1 by binding -> prevents ATP hydrolysis back to APD and Pi
- can drive itself into the F1 subunit complex of the ATP synthase
- regardless of ETC and coupling between the proton transport and ATP synthesis -> if F1 enters the ATP synthase at the alpha beta subunit region the entire system stops
- if you are making more than enough ATP -> you want to avoid the ATP synthase to reverse itself on its own
- F1 subunit can hydrolyze ATP back to ADP by itself
- deprives the cell of its remaining energy resources
- IF1 protects the entire system from hydrolyzing ATP back to ADP and Pi
100
Q
krebs cycle
A
effective biosynthetic pathway
101
Q
tyrosine kinases
A
- insulin receptor
- M2 variant in cancer cells
102
Q
warburg effect
A
- carry out synthesis of lactic acid (homolactic fermentation) even though there is oxygen around
- cancer cells
- yeast
103
Q
oncogenes
A
- p53
- AKT
- entore
- activation of the pathways: aerobic glycolysis, aerobic homolactic fermentation, krebs cycle driven by glutamine and glutamate -> alpha-ketoglutarate, partial activation of krebs to malate and oxaloacetate
- neoplastic transformation
104
Q
fats
A
- cancer cell metabolism
- fatty acid synthase (FASN) tends to be activated
- drugs inhibit fatty acid synthase are antineoplastic reagents
- FASN inhibitors are antiproliferative towards tumor cells
- inhibit proliferative activity
- orlistat (Xenical) inhibits fatty acid synthase and lipases secreted by pancreas and in the stomach
- antilipase activity- weight loss
- antagonism of fatty acid synthase repurposed
- activator of catabolic rxns in cells -> AMP kinase (AMPK) -> phosphorylates
- phosphorylation activates regulated steps in breakdown (glycolysis) and inhibits regulated steps in synthesis
- AMPK sustained activation inhibits tumor growth
- AMPK activates glycolysis by activating PFK-2 (via phosphorylation) -> makes the F2,6diP -> activates PFK-1
- AMPK inhibits acetyl-CoA carboxylase (via phosphorylation) -> which inhibits fatty acid synthesis -> lowers levels of malonyl CoA (rate limiting) > relieve inhibition of carnitine acyl transferase (active) -> enhances fatty acid entry into the mitochondria and breakdown to acetyl-CoA
105
Q
AMPK
A
- catabolic pathway generally
- activated by phosphorylation by liver Kinase B1 (LKB1)
- stimulates a catabolic cascade
- defective AMPK is associated with increase tendency to develop cancer -> mutation in LKB1
- activation of LKB1 -> antiproliferative signal
- activator of catabolic rxns in cells -> AMP kinase (AMPK) -> phosphorylates
- phosphorylates and activates regulated steps in breakdown (glycolysis) and inhibits regulated steps in synthesis
- AMPK sustained activation inhibits tumor growth
- AMPK activates glycolysis by activating PFK-2 (via phosphorylation) -> makes the F2,6diP -> activates PFK-1
- AMPK inhibits acetyl-CoA carboxylase (via phosphorylation) -> which inhibits fatty acid synthesis -> lowers levels of malonyl CoA (rate limiting) > relieve inhibition of carnitine acyl transferase (active) -> enhances fatty acid entry into the mitochondria and breakdown to acetyl-CoA
106
Q
effects of AMPK
A
- in the heart homolactic fermentation is stimulated by AMPK
- in skeletal muscles glycogen storage is diminished
- glycolysis is stimulated
- catabolism in skeletal muscles includes utilization of pyruvate and fatty acids for catabolic rxns -> krebs cycle and CO2
- in liver all the biosynthetic rxns (gluconeogenesis, synthesis of steroids and cholesterol from acetyl-CoA) are inhibited
- ultimate catabolic signaling enzyme
- shuts down ATP consuming biosynthetic rxns and stimulating oxidative catabolic pathways that lead to increase ATP production
- proanabolic and anticatabolic signals (insulin) are inhibited by AMPK
- glycogen synthase is inhibited bc of this
- the source of insulin (beta cells of pancreas) are inhibited by AMPK too
107
Q
AMPK inhibits synthesis of fatty acids, cholesterol, and glucose in liver
A
- AMPK phosphorylates and inhibits acetyl-CoA carboyxlase ACC1 and FS -> FA synthesis decreases
- AMPK phosphorylates and inhibits HMG-CoA reductase -> cholesterol synthesis decreases
- AMPK phosphorylates and inhibits TORC2 -> inhibits transcription of gene coding PEPCK -> turns off gluconeogenesis to conserve ATP
108
Q
AMPK promotes FA oxidation and glucose uptake, but inhibits glycogen synthesis in skeletal muscle
A
- AMPK inhibits ACC -> malonyl-CoA decreases -> lifts inhibition or activates carnitine palmitoytransferase -> more fatty acids move into mitochondria for beta-oxidation
- AMPK increases recruitment of GLUT4 to plasma membrane -> stimulates expression of gene encoding GLUT4 -> facilitates insulin-independent uptake of glucose
- AMPK inhibits G5 -> inhibits glycogen synthesis
109
Q
overview of signal transductions
A
- extracellular small molecule (typically hormone) binds to a receptor
- receptor is coupled to a membrane associated kinase through a g-protein
- g-protein coupled receptors transfer the extracellular signals to the kinases in order to activate them
- 3 common forms of receptor mediated g-protein coupled receptor activations: insulin receptor, adenylate cyclase, phosphoinositide
- g-protein coupled receptors can be activating or inhibitory
110
Q
G-protein coupled receptor: insulin
A
- tyrosine kinase
- leads to inflammation as well as tumor cell invasiveness
- tyrosine kinases are coupled via g-proteins
111
Q
G-protein coupled receptor: phosphoinositide pathway
A
- triggered by generation of breakdown of the phospholipids in phosphatidylinositol
- breakdown of phosphatidylinositol generates a bunch of signals inside the cell -> include inositol triphosphate and residue (diacyl glycerol) of the mechanism by which phosphatidylinositol is incorporated into biological membranes
- when phosphatidylinositol is hydrolyzed diacyl glycerol (DAG) is produced and inositol triphosphate (IP3) (secondary signaling molecules)
- initiation of the hydrolysis of phosphatidylinositol is achieved via g-protein coupled to phospholipase C that breaks it down
112
Q
g-protein coupled receptor: adenylate cyclase
A
- activation of the phosphorylating enzyme: protein kinase A
- receptors detects an extracellular signal
- glucagon or epinephrine are extracellular signaling molecules -> activate protein kinase A
- triggers communication to a heterotrimeric g-protein- associated with protein kinase A signal transduction pathway
- communication is between the receptor of the hormone (glucagon and epinephrine) which activates a membrane bound enzyme -> adenyl cyclase
- adenyl cyclase makes cyclic AMP -> cyclic AMP activates protein kinase A
- product of pathway (cyclic AMP) can destroyed before it activates protein kinase A -> hydrolyze cyclic AMP and convert it to AMP
- enzymes that inhibit phosphodiesterase (hydrolyzes cAMP) are drugs -> Viagra -> vasodilation
- certain toxins that keep the activating g-protein from dissociating -> keep on all the time -> cholera toxin
- certain toxins turn off the activity of the (inhibitory) g-protein coupled receptors that would otherwise be activated -> pertussis
113
Q
adenyl cyclase
A
- membrane bound enzyme that makes cAMP
- stimulates catabolic pathways
- multidomain membrane protein complex
- domains that inhibit adenyl cyclase
- cAMP is capable of activating the protein kinase A
- PKA catalytic subunits are activated by dissociation of regulatory subunits upon binding of 2 cAMP to each regulatory subunit -> displace the catalytic subunits that can now bind protein targets
- protein kinase A can feedback and inhibit adenyl cyclase
- activating g-proteins -> alpha subunit of the g-protein is responsible for activating adenyl cyclase
- prevention of alpha subunit of the inhibitory g-protein from being released to reach adenylyl cyclase -> pertussis toxin -> results in activation of adenyl cyclase
114
Q
activating pathway of adenylate cyclase
A
- receptor for glucagon or epinephrine (specific for activation)
- g-protein (which is heterotrimeric) -> alpha subunit (binds GDP or GTP) and beta and gamma subunits which are always tightly associated
- receptor that responds to the extracellular signal causes dissociation of the g-alpha subunit from the g-beta/g-gamma subunits
- dissociated g-alpha subunit activates or inhibits adenyl cyclase (in this case activate bc glucagon and epinephrine are specific to activation)
- g-alpha is released from g-beta and g-gamma which turns on adenyl cyclase -> cAMP is generated -> protein kinase A is turned on
115
Q
sequence of events for adenyl cyclase activation and deactivation
A
- the sequence of events starts with the heterotrimeric protein associating with the receptor GDP bound to the g-alpha subunit
- heterotrimeric g-protein are capable of shuttling between receptors and adenylate cyclase
- when the receptor interacts with the heterotrimeric g-protein AND a ligand like glucagon or epinephrine -> there will be an exchange with GDP with GTP -> guanine exchange factor rxn
- guanine nucleotides bind to Galpha subunit
- GTP is now bound to g-alpha subunit
- GTPase activity of Galpha shuts down heterotrimeric g-proteins
- GTP bound g-alpha subunit dissociates from the receptor -> diffuses and activates adenyl cyclase
- adenyl cyclase is activated as long as the GTP bound g-alpha subunit is associated
- hydrolysis of the GTP on the g-alpha subunit favors dissociation of the g-alpha subunit -> turns off activation of adenyl cyclase -> activates protein kinase A
116
Q
protein kinase A
A
- activated by adenyl cyclase
- target of cAMP
- hetertetrameric enzyme
- 2 regulatory and 2 catalytic subunits
- favors breakdown (glycolytic and glycogenolytic enzyme)
- catalytic subunits- activates phosphorylase (chemical modification)
- phosphorylase is also subject to allosteric regulation (non chemical)
117
Q
breakdown of glycogen
A
- phosphorylation cascade that favors breakdown of glycogen involves other targets like phosphorylase kinase, cAMP, activating protein kinase A
- activating protein kinase A is a means for phosphorylation phosphorylase and phosphorylase kinase
- protein kinase A- catalytic subunit
- glycogen phosphorylase is activated via protein kinase A
- phosphorylase is regulated by allosteric effectors and chemical modification through phosphorylation
118
Q
liver adapts to changing metabolic condition
A
- portal vein carries nutrients to the liver
- hepatocytes turn nutrients into fuel
- hepatocyte enzymes turn over quickly
- enzymes increase or decrease with changes in diet and the needs of other tissues
119
Q
fates for glucose-6-phosphate in the liver
A
- dephosphorylate to yield free glucose to send to other tissues
- make into liver glycogen
- enter glycolysis, make acetyl CoA and then ATP for hepatocytes themselves
- enter glycolysis, make acetyl CoA to be made into fatty acids and then TAGs
- enter pentose phosphate pathway to yield NADPH and ribose-5-phospahte
120
Q
muscles (myocytes): two types
A
- slow-twitch (red muscle):
- fed by many blood vessels
- rich in mitochondria (to provide energy via slow and steady oxphos)
- fast twitch (white muscle):
- fewer mitochondria and lower O2 delivery
- uses ATP faster and fatigues faster due to greater demands (more tension) combined with reduced O2 delivery
- endurance training can increase mitochondria
121
Q
energy source for muscle contraction
A
- muscle glycogen -> glucose-6-phosphate
- yields 3 ATP, not 2 (as in glycolysis)
- glycogen breakdown skips ATP dependent hexokinase rxn
- pyruvate -> lactate to create NAD+ to enable glycolysis to continue
- phosphocreatine is another energy source
- phosphocreatine + ADP -> burst of heavy activity -> creatine + ATP (other way around during rest)
- acted on by creatine kinase to release ATP
- during light activity or rest- fatty acids, ketone bodies, blood glucose is used
122
Q
hormonal control of glycogen mobilization
A
- epinephrine cascade stimulates glycogen phosphorylase
- break down glycogen in muscle and liver
123
Q
O2 debt
A
- after vigorous exercise, rapid breathing continues
- used for oxidative phosphorylation to build proton gradient and replenish ATP
- ATP used for gluconeogenesis to use up lactate and restore muscle glycogen concentration (cori cycle)
124
Q
the cori cycle
A
- in skeletal muscle that is capable of bursts of activity there is a process that allows the muscle to receive ATP from aerobic mitochondria or anaerobic catabolism that only uses glycolytic path
- during anaerobic activity lactate is produced
- lactate enters the blood and goes to the liver -> uses lactate as a fuel
- uses ATP in gluconeogenesis to make glucose form lactate (during recovery)
- glucose leaves liver and returns to the muscle which uses its own glycogenic pathway to build up glycogen for next period of active contraction
- liver making glucose from lactate
- muscle making lactate from glucose
- there is a version of the cori cycle that works in the heart
125
Q
heart muscle versus skeletal muscle
A
- heart muscle has more mitochondria (50% of cell volume)
- it is fueled primarily by fatty acids (some ketones, some glucose, some phosphocreatine)
- uses fatty acids (preferably) as the krebs cycle substrate
- glycolytic path runs through pyruvate and goes through part of gluconeogenesis to make oxaloacetate and malate -> these are kreb cycle intermediates that can now use the krebs cycle to oxidize acetyl CoA (from fatty acids) all the way to CO2
- it is an aerobic organ
- if the O2 supply is cut off, the muscle dies -> myocardial infarction
126
Q
aerobic organ
A
- makes ATP in mitochondira
- substrate of choice will be fatty acid oxidation
- acetyl CoA is the substrate of the krebs (doesnt prefer)
- acetyl CoA is used for oxidation not used for biosynthetic purposes
127
Q
heart: energy
A
- three major demands: house keeping functions (you need ATP), ion pumping for contraction, contraction (ATP for movement of myosin heads)
- energy demands are a lot
- we must integrate fatty acid catabolism and carbohydrate catabolism to get the energy we need
- getting fatty acids into the mitochondria is the rate limiting step in fatty acid catabolism
- carnitine acyl transferase shuttles the fatty acids into the heart
- once the fatty acids are in they will be catabolized/oxidized all the way to acetyl CoA -> no intramitochondrial regulatory step (this is why regulation at the carnitine acyl transferase is important)
- carbohydrates are converted via the glycolytic pathway to pyruvate
- pyruvate can be completely oxidized to acetyl CoA via pyruvate dehydrogenase however if there is enough acetyl CoA from fatty acid catabolism it can chose not to
128
Q
heart: acetyl CoA
A
- derived from fatty acid catabolism or carbohydrates converted from pyruvate to acetyl CoA via pyruvate dehydrogenase
- fatty acid oxidation mostly is unregulated
- fatty acids is the preferred method of making acetyl CoA
- acyl transferase that make use of carnitine bring fatty acids into the heart -> regulated
- use a little bit of gluconeogenic pathway to make kreb cycle intermediates in order to make full use of the fatty acid derived acetyl CoA
- acetyl CoA inhibits pyruvate dehydrogenase -> fatty acid catabolism shuts down carbohydrate metabolism
- krebs cycle and ETC of mitochondria is supplying the ATP
129
Q
creatine
A
- if there is more than enough ATP the heart stores glucose and uses it to make glycogen
- BUT more importantly it can store creatine
- creatine can be phosphorylated to phosphocreatine
- phosphocreatine has a high energy phosphate bond -> high energy nitrogen bond that can be used to make ATP
- phosphocreatine is hydrolyzed and phosphorylates ADP
130
Q
advantage of acetyl CoA from fatty acid catabolism/oxidation: 2 inhibition methods
A
- two products of fatty acid oxidation that we need to consider: coenzyme A in the form of acetyl CoA and NADH
- both of these products are regulatory towards pyruvate dehydrogenase -> allosteric negative effectors
- fatty acid oxidation inhibits the oxidation of pyruvate via pyruvate dehydrogenase to acetyl CoA
- shut down is not extreme bc the activity of the mitochondria is enough to use up a lot of the NADH in ETC and acetyl CoA in the krebs which slows inhibition
- if krebs and ETC slow down the allosteric negative effectors accumulate and feedback on to pyruvate dehydrogenase
- NADH and acetyl CoA can shut down pyruvate kinase through another mechanism (analogous to shut down of PFK2 and F26biP
- a kinase stimulated by cAMP phosphorylates E1 of pyruvate dehydorgenase -> inhibits
- phosphatase stimulated by insulin removes the phosphate from E1 -> relieves inhibition
- excess NADH also tends to slow the glycolytic pathway at other steps (glyceraldehyde-3-phosphate dehydrogenase step) -> shys away from glycolysis while fatty acid catabolism is going on
131
Q
heart: lactate
A
- heart takes up lactate from skeletal muscle
- extracts lactate from the blood and use it for fuel
132
Q
depletion of O2 to the heart
A
- when we decrease blood and therefore O2 we decrease mitchondrial metabolism which is necessary for fatty acid catabolism -> glycolysis increases which causes accumulation of lactate, protons (lower pH)
- atherosclerotic cardiovascular disease
- blood passes through at a reduced rate (reduced prefusion) -> low O2 and nutrients
- reduced reduction of blood and O2 will compromise hearts ability to wash out metabolic products of contractile activity
- the heart may switch over to some modest anaerobic glycolysis -> uses glycogen stores to supplement reduced glucose form reduced blood flow -> generates lactate
- accumulation of lactate
- protons form glycolysis and mitochondrial metabolism will decrease pH -> negative effect on glycolysis (PFK is pH sensitive)
- if you lower pH the muscle cant carry out glycolytic metabolism as well
- therefore is glycolysis if slowed, and mitochondrial activity is slowed (low oxygen) -> myocardial infarction
- clots will cause serious problems
133
Q
chronic ischemia
A
- people who blood supply is not completely cut off
- function normally by reducing the obligate dependency of the heart on fatty acid catabolism
- partially inhibit the catabolic pathway
- do this by using trimetazidine- partial inhibitor
- trimetazidine inhibits thiolase enzyme (beta-ketoaceyl transferase)
- trimetazidine is a partial inhibitor meaning that fatty acid catabolism is not completely stopped but significantly reduced
- partially reduction of fatty acid catabolism -> NADH and acetyl CoA (products) is partially depleted -> pyruvate dehydrogenase is less inhibited
- heart switches from fatty acid catabolism to glycolytic oxidation (pyruvate dehydrogenase) when under chronic ischemia (slow blood flow)
- glucose and lactate are used as sources of energy (they are not absolutely dependent on O2) -> can use anaerobic when there is less O2
134
Q
diabetes
A
- ketone bodies accumulate in blood (ketosis)
- at risk for elevated levels of acetyl CoA and NADH in the cardiac mitochondria
- endogenously produced acetyl CoA and NADH from fatty acid oxidation combine with the other acetyl CoA NADH cardiac mitochondria -> reduce the ability of the heart to use glycolysis (inhibits pyruvate dehydrogenase)
- ketone bodies are sources of acetyl CoA
- only organ that can make ketone bodies, but also cant use -> liver
- heart can use ketone bodies -> accumulation of acetyl CoA -> poorly perfused (same as chronic ischemia)
135
Q
maintaining angina (chest pain) due to cardiac ischemia
A
- give insulin -> helps allow entry of glucose into cardiac muscle
- insulin also relieves the inhibition of pyruvate dehydrogenase E1 through phosphatase
- improving blood flood -> nitroglycerin (dilates vessels)
- inhibit fatty acid oxidation with drugs like trimetazidine
136
Q
chloroacetate
A
- specific inhibitor of the kinase that inhibits E1 by being phosphorylated
- drug
137
Q
drugs that inhibit the carnitine acyl transferases
A
- reduces the levels of fatty acids entering the heart
- allow the heart to rely more on glycolysis and carbohydrate metabolism
138
Q
resting state
A
-flux through krebs is low bc there is low local concentrations of NAD+ if the rate of oxidative phosphorylation is low
139
Q
why is krebs aerobic even though it doesnt use O2
A
- bc it produces reduced electron carriers that are reoxidized by transferring their electrons to O2
- without O2 electrons build up -> glycolysis starts producing lactate (instead of pyruvate) -> inhibits krebs
140
Q
NADH
A
- cannot transport one electron at a time
- FMN, FAD, ubiquinone, and heme can
141
Q
if ascorbate and methylene blue or TMPD are added to a mitochondria that is inhibited with antimycin a or stigmatellin
A
-about 1 ATP can be made per O2 consumed using the proton gradient established in part by selective uptake of protons from the matrix in a sequence of rxns that result in reduction of oxygen to water
142
Q
citrate synthase
A
-catalyzes an unfavorable rxn that is pushed in the forward direction by the concentration of oxaloacetate
143
Q
isocitrate dehydrogenase
A
- can be found in at least two isozymes
- one in cytosol that use different redox cofactors
- one uses NADPH and the other NADH
144
Q
similarities between PDH and alpha-ketoglutarate dehydrogenase
A
- both employ TPP to convert a carbonyl into a hydroxyl and supply a double bond beta to the carboxyl in order to eliminate it
- both react an aldehyde with the disulfide form of a cofactor to generate a thiohemiacetal
- both release a product from their 2nd subunit, E2, that has a high energy bond
- both use flavin to reduce a nicotinamide
145
Q
GTPase
A
-GTPase activity of alphaG subunit causes it to dissociate from AC -> shuts down cyclase activity
