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Transcript
Reaction mechanism of pyruvate carboxylase acidic anhydride bond biotin carboxylase domain - enzyme biotin transcarboxylase dom. -enzyme carboxybiotin oxaloacetate Pyruvate carboxylase: found in mitochondrial matrix homotetramer prosthetic group: biotin carboxylates pyruvate to oxaloacetate ligase: requires ATP breakdown to ADP + Pi has 3 domains: biotin carboxylase (BC), biotin carrier part (BCCP), transcarboxylase = carboxytransferase (CT) needs Mg2+ as cofactor to bind ATP K+, VO2+ as activators to bind HCO3Mn2+ to maintain enzyme structure acetyl-CoA is an obligate allosteric activator (derived from glucose in well-fed and from fatty acids or ketone bodies in fasting state) Localization of PC high amount in gluconeogenetic: liver and kidney cortex and fat producing tissues: adipose, lactating mammary gland, liver and pancreas beta-cells moderate amount in astrocytes in brain, heart, adrenal gland, small amount: white blood cells, fibroblasts missing from neurons, red blood cells... Wellfed liver cell •glycogenesis (glycogen synthesis) •glycolysis •amino acid degradation •protein synthesis •ornithine cycle •FA and TAG synthesis •PL, SL, VLDL production •cholesterol, its ester synthesis •bile acid and bile production insulin secretion ? NADPH M carbohydrates ↓ ↓ ↓ gal glu fru ↓ ↓ glycogen pyr ↓ PDHC mt.bm. pyr → AcCoA pyruv.carbox. ↓ OA → citr ↑ ↓ NAD ↓ NADH+H+ █ M ←← αKG ADP+P ATP In pancreatic beta cells the high blood and cytoplasmic glucose cc. is the main signal of insulin secretion. Aerobic glucose degradation, high ATP, NADPH etc. production are required cytopl. carbohydrates VLDL lipids bile ↓ ↓ ↓ ↑ ↓ ↓ ↑ gal glu fru TAG,PL,SL←FA cholest. ↓ ↓ glycogen pyr malate ↓ NADPH OA matrix pyr → AcCoA PC ↓ →AcCoA OA → citr O citr ↑ ↓ NAD ↓ NADH+H+ █ M ←← αKG ADP+P slows down ATP 1.) In triacylglycerol synthesizing hepatocytes, white adipocytes, lactating mammarian cells fat is produced mainly from carbohydrates, Pyruvate carboxylase is necessary to increase citrate level, which will be the precursor and allosteric activator of FA synthesis, so PC has an anaplerotic role for citric acid cycle. 2.) Phospholipid (PL) and sphingolipid (SL) synthesis also requires sufficient carbohydrate level normally everywhere. 3.) In brain the FA transport across the blood-brain barrier is not significient, therefore CNS must synthesize its onw fats and can not take it up. In case of PC deficiency the fatty acid, phospholipid, myelin sheat synthesis is impossible causing action potential transmission disturbancies and neurological signs in utero and early in life. 4.) In other tissues both TAG and PL synthesis can be normal, because FA uptake can replenish lipid synthesis from carbohydrates. proteins carbohydrates VLDL lipids bile ↓ ↓ ↓ ↑ ↓ ↓ ↑ amino acids gal glu fru TAG,PL,SL←FA cholest. NH3 ↓ ↓ glycogen pyr malate citr ac c. ↓ AcCoA OA pyr → AcCoA Glu ↓ →AcCoA AS Asp O Asp ← OA → citr O citr ↑ NH3 ↑ ↓ NAD citr citr ↓ NADH+H+ █ M ←← αKG Arg orn orn NH3←↑ ADP+P Glu urea ATP fum mal NH3 ← Gln Anabolic reactions that whithraw intermediets from the citric acid cycle and speeding up the cycle require anaplerotic, filling up reactions, e.g. pyruvate carboxylase, glutaminase, glutamate dehydrogenase. a.) In liver NH3 is biult in and detoxified to urea = carbamide in ornithine = urea cycle. It needs Asp that is derived from oxaloacatete which comes from pyruvate. In pyruvate carboxylase deficiency ammonia is not eliminated good enough, ornithine cycle intermediets are increased: e.g. citrulline. Hyperammonemia is harmful for brain. NH3 is derived from amino acid degradation (from tissues, gut lumen, liver). Amino acid concentration is high after protein rich meal (from gut) and in fasting, when muscle and liver proteins are degraded. b.) malate-aspartate shuttle can export cytoplasmic glycolytic NADH hydrogen to mitochondrial matrix to electron transport chain alpha-ketoglutarate-malate and aspartate-glutamate(+H+) antiporters take part In PC deficiency the NAD/NADH ratio is abnormal, mitochondrial membrane potential is disrupted, mitochondrial structural abnormalities occur. c.) in brain the astrocytes produce glutamine from glutamate, that is derived from a-ketoglutarate. It withraws CAC intermediate. Glutamine is taken up and transformed to glutamate in neurons. Glutamate is the main stimulatory neurotransmitter. Aspartate comes from oxaloacetate, GABA from Glu. Asp and GABA are also neurotransmitters. In PC deficiency these amino acid neurotransmitters can not be produced properly, signal transduction is disturbed. Treatment: - Supplementation of biotin, the prosthetic group. - Aspartate that can take part in urea cycle and fills up citric acid cycle. - Triheptanoin = triglyceride containing 7 carbon fatty acids, that degraded to propionyl-CoA, converted to succinyl-CoA filling up CAC in most cells. In liver 5 C containing ketone bodies are produced, transported to CNS and fill up the citric acid cycle there, improving all the metabolic and neurological disturbances. Fasting in liver • • • • • • • glycogenolysis = glycogen degradation gluconeogenesis amino acid degradation urea cycle FA beta-oxidation ketone body production (VLDL production and glyceroneogenesis from lactate or amino acids) adip. TAG ↓ glycerol ↓ glyc3P brain, rbc, heart ↑ glucose ↑ glycogen DHAP PEP ↑ OA fum → mal Arg urea rbc, white muscle ↓ lactate ↓ amino acids pyruvate pyr PC↓ Asp → OA → P citr ← citr orn → orn ← AcCoA citr ↓ ↓ M ← ← αKG ↑ ↑ amino acids NH3 liver and muscle proteins C atoms of lactate, glycerol, amino acids are built into glucose N atoms of amino acids are built into urea 1.) Pyruvate carboxylase is one of the regulated enzymes of gluconeogenesis, if the precursors are lactate, and amino acids degraded to pyruvate. In liver and kidney cortex gluconeogenetic regulated enzymes are - induced by adrenalin, noradrenalin leading to cAMP elevation, - by cortisol - repressed by insulin. In PC deficiency gluconeogenesis is not efficient causing hypoglycemia, which is wrong for the obligate glucose consuming brain, red blood cell, white skeletal muscle, kidney medulla etc. Pyruvate is converted to lactate and alanine in the cytoplasm, causing lactic acidosis, Metabolic acidosis deteorates brain. 2.) Low blood glucose level causes glucagon secretion, causing FFA increase, leading to ketone body production i.e. ketoacidosis. adip. TAG ↓ glycerol ↓ glyc3P brain, rbc, heart ↑ glucose ↑ glycogen DHAP PEP ↑ OA Arg urea rbc, white muscle ↓ lactate ↓ amino acids pyruvate ketone bodies pyr ↓ Asp → OA → P fum → mal brain, muscles citr ← citrul. orn → orn ← AcCoA ←← FA-CoA citrate ↓ ↓ M ← ← αKG ↑ ↑ amino acids NH3 liver and muscle proteins adip. TAG FFA FA cytolasm mitoch matrix Pyruvate carboxylase deficiency – group I. point mutations: Val145 → Ala, or Arg451→Cys in biotin carboxylase domain → enzyme acitivity↓↓ in North Amariacan native people signs: lactic acidemia (metabolic acidosis) mild to moderate (pyr and Ala ↑, too) delayed development psychomotor retardation Pyruvate carboxylase deficiency group II. point mutation: Ala610 →Thr or Met743 →Ile in transcarboxylase domain → protein instabilty and degradation → missing PC enzyme in UK and French patients signs: serious lactic acidemia, Pyr and Ala are high improper gluconeogenesis causes hypoglycemia oxaloacetate ↓→ Asp ↓→ urea cycle in liver ↓→ NH3 is not detoxified ↓→ brain damage and citrullin ↑ in blood anaplerotic reac. of citrate cycle ↓→ Citric ac. cycle ↓→ ATP ↓→ brain damage Regulation of pyruvate carboxylase glucose → ? → P2= distal promoter activated pyr. carb. transcription → ? → insulin secretion PPAR → P1= proximal promoter activated → pyr. carb. gene transcription → lipogenesis glucagon, adrenalin... → CREB → P1 promoter activated → PC expression → gluconeogenesis