nov 21 2017.ppt [kompatibilitätsmodus] · triple h-syndrom: hyperornithinemia, hyperammonemia,...
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Gerhild van Echten-Deckert
Tel. 73 2703E-mail: [email protected]
Nitrogen FixationUrea Cycle
Biochemistry
Metabolism
07.11.2017 – 27.11.2017
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Nitrogen metabolism
� N2 assimilation via reduction to NH3 (nitrogenase complex)� NH3 metabolism: glutamate-dehydrogenase
glutamate synthaseglutamine synthetaseglutamine amidotransferase
� urea cycle� C1 metabolism (PLP, THF, SAM, homocysteine)� nucleotide metabolism: biosynthesis of purines and pyrimidines
from RNA to DNA (NDP reductase)salvage pathway, HGPRT deficiencycytostatic drugscatabolism (ADA-deficiency, urate)
Signal transduction
� GPCR – glucagon signalling� RTK – insulin signalling� G-proteins
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Soluble biologically utilisable nitrogen components are generally scarcein natural environments.Thus most organisms maintain strict economy in their use of ammonia, amino acids and nucleotides. These components are often salvaged and reused.
Unlike carbohydrates and lipids, amino acids and nucleotides are not storedin cells. Thus the amount of free amino acids and nucleotides is low and their metabolism is accurately controlled and regulated. These molecules are charged thus affecting the electro-chemical balance in the cell.
Characteristics of Nitrogen Components
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The Nitrogen Cycle
1015t(99%)
KlebsiellaAzotobacterRhizobium
NitrosomonasNitrobacter
Pseudomonas
„Nitrate-respiration“2 NO3
¯ + 10 e¯ + 12 H+ → N2 + 6 H2O
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The Anammox Reactions
Brocadia anammoxidans(Planctomycetes)
1960s/1970s:Anammox: anaerobic ammonia oxidation using nitrite as e—acceptorMid 1980s:Bacteria promoting anammox discovered in Delft in a waste-treatment system.
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Ladderane Lipids of the Anamoxosome Membrane
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Nitrogen Fixation by the Nitrogenase Complex
Nitrogen-fixing nodules Leguminous plant – leghemoglobinBacteria – nitrogenase complex
(oxygen lability)
Dimer, Fe-S30 sec half-life in presence of O2
Tetramer, Mo-Fe-S10 min half-life in presence of O2
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Glutamine synthetase:
Glu + ATP γ -glutamyl phosphate + ADP
γ -glutamyl phosphate + NH4+ Gln + Pi + H+
_____________________________________________________________
Glu + NH4+ + ATP Gln + ADP + Pi + H+ (a)
Glutamate synthase (not present in animals):
α-KG + Gln + NADPH+ H+ 2 Glu + NADP+ (b)
(a) + (b):α-KG + NH4
+ + ATP + NADPH Glu + NADP+ + ADP + Pi
Glutamate dehydrogenase:
α-KG + NH4+ + NADPH+ + H+ Glu + NADP+ + H2O (KM(NH4+ ) = 10mM)
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The sequence of reactions catalyzed by glutamate synthase.
Page
103
1
Biochemistry, Voet & Voet, 3rd ed.
Glu-DH
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Allosteric and CumulativeFeedback Regulation ofGlutamine Synthetase
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Regulation of bacterial glutamine synthetase by covalent modification
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103
5
Biochemistry, Voet & Voet, 3rd ed.
PII: regulatory protein
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Regulation of bacterial glutamine synthetase by covalent modification
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Proposed Mechanism ofGlutamine Amidotransferases
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Overview of catabolism of amino groups in vertebrate liver
1
2
3
4
1: Transaminase2: Transaminase3: Glutaminase4: Glu‐DH
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Urea cycle&
reactions feedingamino groups into
the cycle
Carbamoylphosphate synthetase 1 (CPS)
1. Ornithine transcarbamoylase (OTC)2. Argininosuccinate synthetase (AS)3. Argininosuccinate lyase (AL)4. Arginase (A1)
NH3 from intestinvia vena porta
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Nitrogen-acquiring reactions in the synthesis of urea
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Short-time (allosteric)regulation of the urea cycle
Long-time (rate of expressionof the 5 enzymes of the cycle)regulation of the urea cycleis diet-dependent.
(NAGS)
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Urea cycle disorders
Triple H-Syndrom: Hyperornithinemia, Hyperammonemia, HomocitrullinuriaDefect in Orn mitochondrial transport (no Orn recycling); symptoms: developmental delay, mental retardation, vomiting, ataxia, lethargy, irritability, coma.
Fearing & Shih, Encyclopedia of Biol Chem IV
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Location Abb. Enzyme Disorder Measurements
Mitochondria NAGS N-Acetylglutamatesynthase
N-Acetylglutamatesynthase deficiency +Ammonia
Mitochondria CPS1 Carbamoyl phosphatesynthetase I
Carbamoylphosphate synthetaseI deficiency
+Ammonia
Mitochondria OTC Ornithinetranscarbamoylase
Ornithinetranscarbamoylasedeficiency
+Ornithine, +Uracil, +Oroticacid
Cytosol AS Argininosuccinic acidsynthetase
AS deficiency orcitrullinemia +Citrulline
Cytosol AL Argininosuccinaseacid lyase
AL deficiency orargininosuccinicaciduria (ASA)
+Citrulline, +Argininosuccinicacid
Cytosol A1 Arginase Arginase deficiencyor argininemia +Arginine
The six urea cycle disorders
Inherited autosomal recessive disorders except the X-linked OTC deficiency. Similar clinical features: Hyperammonemia, respiratory alkalosis, seizures, acute encephalopathy, coma, death. Unique to AS deficiency: short friable hair, liver fibrosis
Fearing & Shih, Encyclopedia of Biol Chem IV
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triggers
triggers
Gly-synthase
Gln-synthetase
Therapeutic strategy to overcome deficiencies in urea cycle enzymes
Administration of aromatic acids(benzoat or phenylbutyrate) inthe diet lowers blood ammonia level.
Removal of Gly and Gln triggers NH3-consumingreactions, thus loweringits blood content.
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Links between the urea cycle and the citric acid cycle