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14 Cards in this Set

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Indicate how pyruvate is transported from the cytosol into the mitochondrial matrix.
Glycolysis occurs in the cytosol while oxidation of pyruvate (via the TCA cycle) occurs in the mitochondrial matrix. The inner mitochondrial membrane is impermeable to pyruvate; pyruvate translocase uses a symport mechanism to transfer pyruvate into the membrane with H+ ions. For each pyruvate that enters the mitochondria matrix (4H+/1ATP, thus transport of pyruvate costs ¼ ATP).
Summarise the overall reaction catalysed by the PDH complex.
In order for pyruvate to enter the TCA cycle, it must first be oxidized to acetate in the form of acetyl CoA. This reaction is an oxidative decarboxylation and is thermodynamically irreversible. The oxidative decarboxylation of pyruvate to acetyl CoA commits the C atoms of glucose to either oxidation to CO2 by the Krebs cycle of incorporation into lipid. PDH contains three enzymes: pyruvate dehydrogenase, transacetylase, dihydolipoyl dehydrogenase.
Outline the multienzyme nature of PDH complex and the cofactors that it requires (TPP, Lipoic acid, FAD)
Pyruvate dehydrogenase (decarboxylase) is E1. It has a thyiamine pyrophosphate (TPP) prosthetic group; mammals can’t synthesize thiamine, must be provided in diet as vitamin B1. This enzyme oxidatively decarboxylates pyruvate to the hydroxyethyl group and CO2 which remains bound to TPP.
Transacetylase is E2 and catalyzes the oxidation of the hydroxyethyl group into acetate and its transfer to coenzyme A (CoASH). It contains lipoic acid and provides the core around which other subunits are arranged. Each molecule of lipoic acid is covalently connected through an amide bond to a side chain of Lys in the polypeptide, together known as lipoamide. The long lipoamide arm enables the ring to swing from one active site to another. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (SH) groups in lipoamide, required reoxidation to the disulfide (SS) form to regenerate lipoate as a competent acyl acceptor.
Dihydrolipoyl Denhydrogenase is E3 and catalyzes the regeneration of the oxidized form of lipoamide with concomitant reduction of NAD+ to NADH. In mammals it has six identical chains each bound to a FAD+ molecule as a prosthetic group (strongly bound). The final activity of the PDH complex is the transfer of reducing equivalents from the FADH2 of the dihydrolipoyl dehydrogenase to NAD+.
Explain in outline the regulation of the PDH complex (role of NADH, acetyl-COA and phosphorylation).
The complex is inhibited by its products NADH and acetyl-CoA. In addition, the complex is active in the dephosphorylated state (insulin), but inactive in the phosphorylated state (glucagon); PDH kinase thus inactivates E1 while PDH phosphataste activates E1. Both kinase and phosphatase are bound to the PDH complex. PDH kinase is active when the cellular energy charge is high (ATP, NADH, acetyl CoA); these molecules act as allosteric activators of the kinase and negative allosteric effectors of the phosphatase (E already present, no need to make more). Conversely, pyruvate acts as an inhibitor of PDH kinase and an activator of phosphorylase. The ratios of NADH/NAD+ and acetyl-CoA to CoASH determine activity of the enzymes. PDH phosphorylase is activated by Mg2+ and Ca2+; ATP and citrate are good chelators (bind tightly, remove from solution) of these ions making them unavailable for phosphorylase. As ATP is consumed, the ions are released into solution allowing the PDH phosphatase to once again become activated.
In adipose tissue, the activity of PDH is stimulated by insulin (via PDH phosphatase). In heart tissue, the activity of PDH is stimulated by catecholamines (epinephrine and adrenaline).
Indicate the inhibitory effects of trivalent arsenic compounds on PDH
Trivalent arsenic reacts with reduced limpamide and prevents it from continuing to participate in the transacetylase reaction (hydroxytheyl-TPP into acetyl-CoA). This inhibition can be removed by 2,3 dimercaptopropanol/BAL/Dimercaprol; BAL is also used to treat Hg2+ and Pb poisoning. In WW2, BAL was used as an antidote to Lewisite, arsenic containing war gas. BAL contains two reduced sulphydryl groups (similar to lipoamide) and can consequently compete with lipoamide for the inhibitor. BAL binds to the inhibitor and forms a complex that can then be excreted through the urine.
Discuss the consequences of thaimine deficiency (Wernick-Korsakoff syndrome, Beri-Beri)
Thiamine (Vit B1) is required to make TPP (a prosphetic group in PDH-E1, alpha-ketoglutarate [TCA], and transketolase [PPP]). Deficiency in this enzyme causes the condition known as Beriberi. This disease is more common in Asia than in the west because Asian diets often lack thiamine (lost on the husks of rice, but present in meat and yeast). A minor deficiency results in abnormal sensations in the limbs, paralysis, and muscle wasting (dry beriberi); a severe deficiency gives the same neurological problems, but because the heart is starved of E because of a defective PDH, congestive heart failure can develop resulting in edema (wet beriberi).
In developed countries, thiamine deficiency is most often seen in chronic alcoholics because of poor nutritional habits. Wenicke’s encephalopathy results with neurological symptoms (acute mental disturbances and ocular paralysis). If not treated, coma and death can also occur from sudden cardiac failure.
Summarize the congenital abnormalities affecting the action of the PDH complex and their clinical consequences.
Severe defects in any of the enzymes in the PDH complex will lead to an early appearance of neurological diseases and are often fatal before the first year of life. If the child survives, he/she will be mentally retarded and possibly microcephalic. In such conditions, the levels of pyruvate, lactate, alanin, and alpha-ketoglutarate in the blood are elevated. Pyruvate accumulates in the blood because it cannot be converted into acetyl-CoA and passed onto the TCA cycle (normal aerobic fate). Instead the cell acquires E through the anaerobic glycolysis, resulting in the buildup of lactate. The most common causes of lactic acidosis are defects in the PDH complex, but severe forms of the condition are called Leigh’s disease. Alanine is made from pyruvate by a transamination reaction catalyzed by alanine aminotransferase. Alpha-ketoglutarate can also accumulate because the alpha-ketoglutarate dehydrogenase (TCA) is unable to complete its oxidative decarboxylation reaction. Although these organic acids occur in the body in the ionized form, their precursors are made from free acids; the acids must then be neutralized by buffering systems of the body (HCO3). Thus defects causing the accumulation of these molecules often result in lactic acidosis.
A common form of PDH deficiency is caused by a defect in E1 (gene found on X-chromosome). PDH supplies E to the brain; mutations in the gene affect both males and females, even though females are just carriers (x- linked dominant). Some patients only have a partial deficiency in the activity of the PDH complex; if 30-50% remains asymptomatic until age 5-15 after which there slow progressive ataxia develops. Although there is no cure for PDH deficiencies, a ketogenic diet (high in fats, low in carbs) is beneficial for some because it promotes ketone (produces acetyl-CoA) body synthesis as a source of fuel in the brain.
Explain the central role of the TCA cycle in metabolism, including both its catabolic and anabolic functions(amphibotic role)
The TCA cycle is a cyclic metabolic pathway that is used by aerobic cells for the oxidation of acetate groups to CO2 and production of reducing equivalents in the form of NADH and FADH2. The TCA cycle is the final common pathway for the oxidation of fuel molecules: carbohydrates, FAs, and AAs. In its catabolic role, the TCA cycle is involved mainly in the oxidation of acetyl CoA; however, anabolically, intermediates of the TCA cycle are important in the synthesis of many compounds (AA, heme, glucose). All enzymes except succinate dehydrogenase are located in the mitochondrial matrix (sucinate dehydrogenase is on the inner mitrochondrial membrane with the ETC).
Outline the intermediates and the enzymes of the TCA cycle
X
Explain the energetics of the cycle, including reactions where reducing equivalents (NADH and FADH2) are produced and the production of GTP by substrate level phosphorylation.
Iso-citrate dehydrogenase
NAD+ → NADH, H+ ____________________(3 ATP)
Alpha-ketoglutarate
NAD+ →NADH, H+_____________________(3 ATP)
Succinyl CoA synthetase/thiokinase
GDP → GTP → ATP (nucleoside diphosphokinase)
Succinate Dehydrogenase
FAD+ → FADH2________________________(2 ATP)
Malate Dehydrogenase
NAD+ → NADH, H+_____________________(3 ATP)
Explain why aerobic metabolism of glucose using the TCA cycle is much more efficient than anaerobic glycolysis
The complete aerobic oxidation of one molecule of glucose to CO2 and H2O (using glycolysis, PDH complex, TCA cycle, ETC, and ATP synthase) produces 32-38 molecules of ATP. The anaerobic oxidation of one molecule of glucose produces 2 ATP. (This assumes malate-aspartate shuttle, if glycerol phosphate was used, only 30-36 ATP would be produced). Cycle will only operate during aerobic conditions even though oxygen is not directly used; in its absence NADH and FADH2 cannot be reoxidized and the enzymes using them cannot function.
Explain the regulatory mechanisms for the TCA cycle. Indicate the effect of NADH, ATP, succinyl-CoA, Ca2+, and oxaloacetate
The rate of the cycle is closely linked to the rate of the ETC and both are regulated by ATP
High ATP/ADP levels (high E state)  low ETC/high TCA
Low ATP/ADP levels (low E state)  high ETC/low TCA.
3 Irreversible Steps:
Citrate synthase: Citrate is a competitive inhibitor of oxaloacetate binding to citrate synthase, thus as citrate concentrations increase the activity of citrate sythase decreases. Oxaloacetate is an activator of citrate synthase; the concentration of OAA can increase in the cell from pyruvate via pyruvate carboxylase.
Isocitrate dehydrogenase: Rate limiting step, most important control site. Stimulated by ADP (positive allosteric effector); inhibited by ATP (negative allosteric effector). NADH competes with NAD+ for the binding to isocitrate dehydrogenase, thus when NADH levels are high or when oxygen becomes limited, the enzyme slows down. Ca2+ ions stimulate isocitrate dehydrogenase, serving as a stimulus for the contraction of both skeletal and smooth muscle. In addition, Ca2+ stimulates glycogen breakdown through glycogen phosphorylase.
Alpha-ketoglutarate dehydrogenase is inhibited by succinyl CoA (product) because it is a competitive inhibitor of CoA-SH binding to alpha-ketoglutarate dehydrogenase. NADH is a negative allosteric inhibitor. Ca2+ ions stimulate the enzyme for the same reason as above. If the oxygen supply is limited than the rate of TCA is decreased because of the shortage of NAD+
Indicate the effect of fluorocitrate and malonate on the TCA cycle.
Fluoroacetate (not itself toxic)  fluroroacetyl CoA (via acetyl-CoA synthase)  fluorocitrate (via citrate synthase). Fluorocitrate is a potent inhibitor of aconitase; the F atom of fluorocitrate binds to the Fe2+ ion present in the active site of aconitase. Present in certain toxic plants and rat poison.
Malonate is a structural analog of succinate and acts as a competitive inhibitor of succinate dehydrogenase. It can bind to the enzyme but not react. This is due to the very close structural similarity between malonate and sucinate. Malonate is used experimentally as a very effective inhibitor of the TCA cycle.