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SGU: TCA & ETC
Biochem: TCA Cycle and ETC
| Question | Answer |
|---|---|
| What are the reducing molecules used in the ETC to make ATP? | NADH, FADH2 |
| Where are the enzymes of the PDH and TCA cycle found? | pyruvate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, in the mito matrix |
| Which membrane of the mito is leaky, which is the major barrier? | outer: leaky, inner:barrier |
| How does pyruvate enter the mito? | pryuvate translocase transports pyruvate across the inner mito membrane in symport with H+ |
| WHat converts pyruvate to Acetyl CoA? | the pyruvate dehydrogenase complex does and produces NADH in the process |
| Describe the PDC | 3 enzymes: E1:pyruvate dehydrogenase, E2:dihydrolipoyl transacetylase, E3:dihydrolipoyl dehydrogenase and 5 co-enzymes |
| What products/coenzymes are required for the action of PDH? | pyruvate, CoA-SH, NAD+, TPP, lipoate, FAD |
| Describe the properties of Acetyl-CoA | high energy compound, hydolysis of the thioester is very high (greater than ATP hydrolysis) |
| Functions of Acetyl-CoA | input into TCA cycle where the acetate moiety is further degraded to CO2, donor of C2 for synthesis of fatty acids, ketone bodies, cholesterol, amino acids |
| Why is there such a large complex of enzymes for the PDC? | enzyme complex can direct substrate from one enzyme to the next, channeling intermediates minimizes side reactions, the reactions can be coordinately controlled |
| What is the prosthetic group of Pyruvate dehydrogenase? | thiamine pyrophosphate (TTP) |
| What is the prosthetic group of Dihydrolipoyl transacetylase? | lipoamide |
| What is the prosthetic group of dihydrolipoyl dehydrogenase? | FAD |
| What is the function of E1? | pruvate is decarboxylated to form hydroxyethyl derivative bound to TPP |
| Describe the function of E2? | hydroxyethyl is oxidized by transfer to the disulfide form of lipoic acid (E2 prosthetic), the acetyl group bound as a thioester to the side chain of lipoic acid is transferred to CoA |
| Describe the function of E3? | sulfhydryl form of lipoic acid oxidized by FAD-dependent E3 leading to the formations of oxidized lipoic acid making FADH2. FADH2 is oxidized by E3 as NAD is reduced to NADH |
| What are all the vitamens in the PDC? | panthothenic acid:coenzyme A, Thiamine:TPP, lipoic acid:lipoamide, riboflavin:FAD, Niacin:NAD |
| What are the prosthetic groups of PDC? | TPP, FAD, lipoamide |
| What are the cosubstrates of PDC? | CoA, NAD |
| Describe Beriberi | nutritional deficiency of thiamine, especially affects the brain because of it's dependence on glucose metabolism, prolonged deficiency causes multiple system effects, occurs with alcohol abuse |
| What is Wernicke-Korsakoff? | Also known as wet brain, thiamine deficiency due to alcohol abuse |
| What is genetic beriberi? | ppl who can't absorb thiamine |
| How is the PDC inhibited? | NADH competes with NAD+ for binding to E3, Acetyl CoA competes with CoA for binding to E2 |
| How is the PDC activated? | by NAD to E3 and CoA to E2, phosphorylation state of E1, PDH kinase/PDH phosphatase |
| How many copies of each enzyme are there per complex? | E1:20-30, E2:1, E3:6 |
| How is E1 regulated? | PDC kinases catalyze E1 phosphorylation thereby inhbiting it |
| How are the PDC Kinases activated? | they are activated by NADH and acetyl CoA, PDH kinases interact with E2 to sense changes in oxidation state and acetylation of lipoamide by NADH and acetyl CoA |
| How do PDH kinases work during the fasted state? | in muscle PDH kinase increases which inhibits msucle from using glucose that is more needed for brain |
| How is PDH kinase inhibited? | in the presence of high pyruvate concentration it is inhibited by ADP |
| What is the function of PDH phosphatases? | removes Pi from E1, in skeletal muscle it is activated by Ca allowing mito metabolism to be stimulated during exercise |
| What is arsenite? | trivalent arsenic |
| How does it regulate PDH? | forms a stable complex with lipoic acid and inhibits the E2 enzyme causing lactate to accumulate and a significant reduction of energy |
| Describe Leigh syndrome | inherited disorder that affects infants caused by mutation in PDH complex resulting in reduced ATP production. Degradation of motor skills and episodes of lactic acidosis |
| Describe E2 enzyme deficiency | hyperammonemia, non-specific amino acid elevation, discrete lesions on the globus pallidus |
| Describe E3 enzyme deficiency | branched chain amino acid elevation in serum, alpha ketoglutarate in serum and urine, TPP inhibitors can be detected in urine and blood |
| What reactions replenish intermediates of the TCA cycle? | anapleurotic reactions |
| What does the amphibolic nature of the TCA cycle refer to? | anabolic reactions to generate energy and anabolic reactions that generate intermediates for biosynthesis occur in the TCA cycle |
| What kind of intermediates are in the TCA cycle? | carbohydrates, lipids, amino acids, as well as nucleotides and porphyrins |
| What is produced in the TCA cycle? | 3NADH, 1FADH2, 1GTP |
| How many ATP produced when glucose is fully oxidized to CO2 and H20? | (34) 36 |
| How many ATP are contributed by the TCA cycle and by which coenzyme? | 24 ATP in total, 2 ATP by substrate level phosphorylation, 18 ATP from 6 NADH, 4 ATP from 2 FADH2 |
| which enzyme in the TCA cycle catalyzes the highest free energy reaction? | citrate synthase |
| Which enzyme in the TCA cycle catalyzes the lowest free energy reaction? | malate dehydrogenase |
| 1st step of TCA? | Condensation of Acetyl CoA with oxaloacetate produces citrate, reaction is irreversible, catalyzed by citrate synthase |
| Properties of citrate? | can be exported out of the mito, can be broken down by ATP-citrate lyase to yield oxaloacetate and acetyl-CoA, it inhibits PFK1 and activates acetyl-CoA carboxylase of fatty acid synthesis |
| Properties of citrate synthase? | Oxaloacetate binds citrate synthase, conformational change generates acetyl-CoA Inhibited by citrate, NADH, succinyl-CoA, ATP. Activated by ADP |
| What happens to citrate in the TCA cycle? | isomerized by aconitase to isocitrate |
| Inhibition of aconitase? | inhibited by fluoroacetate |
| Where is fluoroacetate found? | compound in rat poison |
| Describe the action of fluoroacetate. | converted to fluoroacetyl CoA and condenses with oxaloacetate to form fluorocitrate which inhibits aconitase causing citrate build up |
| What happens to isocitrate in TCA cycle? | irreversible reaction, catalyzed by isocitrate dehydrogenase, produces alpha-ketoglutarate and NADH and CO2 |
| Describe alpha ketoglutarate | transaminated to make glutamate which can be used to synthesize other amino acids or donate nitrogen groups to other syntheses |
| Regulation of isocitrate dehydrogenase? | inhibited by ATP and NADH, activated by Ca2+, ADP |
| What are the functions of the two forms of isocitrate dehydrogenase? | one uses NAD and the other uses NADP, ONLY THE NAD linked enzyme is used in the TCA cycle |
| What is the reaction catalyzed by alpha-ketoglutarate dehydrogenase? | production of succinyl CoA, NADH, and CO2 from alpha-ketoglutarate |
| Regulation of alpha-ketoglutarate dehydrogenase? | inhibited by succinyl CoA, NADH and activated by Ca |
| What is special about the reaction by alpha-ketoglutarate dehydrogenase? | second oxidative decarboxylation producing a very high energy thioester, it is a key regulatory step, similar to the PDC: E1, E2 and E3 enzyme units |
| What is the main difference between PDC and alpha-ketoglutarate dehydrogenase? | not affected by reversible protein phosphorylation, NB: it can be affected by thiamine deficiency |
| What happens to succinyl CoA in the TCA cycle? | cleavage of high energy thioester bond to produce GTP, catalyzed by succinyl-CoA synthetase |
| Describe the transfer of the phosphate to GTP by succinyl-CoA synthase | succinyl CoA has a thioester bond with very negative free energy when hydrolyzed which is transferred to phosphoester bond with inorganic phosphate. The phosphate is transferred to histidine of enzyme and then GDP |
| Succinate reaction in TCA? | succinate is converted to fumarate when it is oxidized by succinate dehyrogenase, produces FADH2 |
| Describe succinate dehydrogenase | located in inner mito membrane, inhibited by malonate which is similar to succinate and competitively inhibits it. Malonate is a metabolic inhibitor and decreases ATP levels |
| Properties of Fumurate | used to make amino acids and pyrimidine nucleotides, it is also made in the urea cyce during purine synthesis and catabolism of certain amino acids |
| Fumurate in the TCA cycle? | fumarase converts it to Malate, reaction is reversible |
| Malate in the TCA cycle? | oxidation of malate to produce oxaloacetate + NADH, catalyzed by malate dehydrogenase |
| Malate dehydrogenase forward reaction has a positive delta G, how does it proceed forward? | there is a lot of substrate and the product, oxaloacetate is constantly being removed |
| Properties of oxaloacetate? | also produced by transaination of aspartic acid, can be used to make amino acids and pyrimidines |
| Regulation of TCA cycle? | high ATP=negative feedback, first 3 steps inhibited by high [ATP], reactions with high negative delta G's are regulated |
| Which are the rate controlling enzymes of the TCA cycle? | citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase |
| NADH & FADH2 are reducing or oxidizing agents? | strong reducing agents |
| What is the order of enzymes in the ETC from high redox potential to low? | NADH-CoQ reductase, succinate-CoQ reductase, CoQH2-cytochrome c reductase and cytochrome c oxidase |
| Why is energy transfer from glucose to ATP not 100% efficient? | because some of the energy is consumed in other reactions such as Ca2+ transport or released as heat |
| How much ATP does a regular body synthesize per day? | 50 grams |
| What are the electron carriers of the ETC? | NADH, FADH2, coenzyme Q, cytochrome C, O2 |
| Function of electron carriers? | move electrons from complex 1-4 until combining with oxygen |
| How many domains does the ATP synthase/complex 5 have? | F0 in the inner membrane and F1 in the matrix |
| What is the function of NADH Dehydrogenase? | takes the proton and hydride ion carried by NADH |
| Is NADH tightly associated with their proteins? | NO, loosely associated |
| Function of NADH? | water-soluble electron carriers |
| Under aerobic conditions what regenerates the NAD from glycolysis reactions? | aspartate-malate and glycerophosphate shuttles |
| Describe the aspartate malate shuttle | NADH reduces OAA in the cytosol to make malate which is moved into the mito matrix by alpha-ketoglutarte-malate transporter. In the matrix malate reconverted to OAA then transaminated to aspartate which is transported back into cytosol |
| Describe the glycerophosphate shuttle | oxidizes NADH converting it to DHAP to G3P, G3P transfers electrons to FAD to make FADH2 in the inner mito membrane, FADH2 transfers electrons to make 2ATP |
| Complex I:NADH Dehydrogenase action | FMN (riboflavin) accepts electrons and protons from NADH+H to become FMNH2, FMNH2 contains an iron/sulphur center which takes two hydrogen and transfers them to coenzyme Q = QH2 (4 protons transferred into intermembrane space of mito) |
| How are flavin derivatives reduced? | by sequential addition of 2 hydrogen atoms (2 e- and 2 p+) |
| How does coenzyme Q/ubiquinone carry electrons? | reduced to ubiquinol from 2 electrons |
| HOw many electrons does semiquinone carry? | 1 |
| HOw many redox states are there is coenzyme Q? | 3 |
| Action of complex II:succinate dehydrogenase | electrons passed from succinate to FAD in TCA cycle, electrons are transferred from FADH2 to three Fe-S centers and then to Q, two protons are taken up to from QH2 |
| Describe cytochromes | contains a heme group that fluctuates between Fe3+ and Fe2+, on the outer face of the inner membrane and is mobile |
| How do the cytochromes bc1 and c/a+a3 receive electrons? | from CoQ |
| Describe the reactions at cytochrome C oxidoreductase/complex III | Q bind CytoB and heme accepts e-, e- transferred to CytoC1, CytoC1 (membrane bound) transfers e- to CytoC (water soluble/mobile) which can diffuses and donate it's electrons to complex IV, 4 protons pumped |
| Complex IV/cytochrome oxidase reaction | iron atoms of the heme groups in the cytochromes and copper atoms are oxidized and reduced as electrons flow from cyto C to O2 to make water, 2 protons pumped |
| When are ROS produced during the ETC? | when O2 is not fully reduced to water but instead produces a ROS:O2-, H2O2, OH- |
| Where does superoxide USUALLY come from? | leaks from complex I |
| What is the effect of ROS on the cell? | DNA damage, membrane damage, oxidation of amino acids, inactivation of enzymes, initiation of apoptosis signalling |
| What enzymes destroy ROS? | superoxide dismutase, catalase |
| What is the free-radical theory? | oxidateive damage initiated by ROS is a major contributor to the functional decline that characterizes aging |
| What occurs during Alzheimer's disease? | degenerative disease caused by accumulation of oxidative damage much faster than would occur naturally, patients have a deficiency in the terminal complex of the mito ETC at complex IV, inherited forms are autosomal dominant |
| How do you identify the location of ETC inhibition? | look at redox states of different complexes, carriers before block are reduced and those after are oxidized |
| Which ETC inhibitors block NADH dehydrogenase/complex I? | amytal and rotenone, piericidin (same mechanism as antimycin) |
| Which ETC inhibitors block complex III and by what mechanism? | antimycin A, preventing the oxidation of ubiquinol |
| Which ETC inhibitors block complex IV and by what mechanism? | cyanide, carbon monoxide, sodium azide, bind to heme groups of the cytochromes, hydrogen sulfide - binds Fe2+ in heme groups |
| Describe amytal | barbiturate: CNS drug, acts on GABA sensitive ion channels enhancing GABA concentrations in the brain, used for insomnia-ideally causing drowsiness |
| Describe rotenone | insecticide: used to catch fish, high doses cause impaired ETC effects such as increase in ROS, decrease in ATP and cell death |
| Describe carbon monoxide affect on cytochrome oxidase | inhibits it |
| Describe cyanide | reversible inhibitor of cytochrom oxidase, binds to Fe3+ heme group |
Created by:
mnoronha
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