7.AA_Metabolism - 21. Amino acid metabolism: nitrogen...

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Unformatted text preview: 21. Amino acid metabolism: nitrogen fixation, 21. transamination and NH3 transport 1 amino acids & other organic compounds Nitrogen cycles between oxidized & reduced forms in the biosphere synthesis (microorganisms, plants & animals) reduction (plants & some anaerobic bacteria) degradation (animals & microorganisms) denitrification nitrate NO3- N2 (anaerobic bacteria) nitrification (Nitrobacter & other soil bacteria) more oxidized nitrogen fixation (Rhizobium & some other bacteria) NO2nitrite NH4+ ammonium nitrification nitrification ( Nitrosomonas & other soil bacteria; marine archaebacteria) more reduced 2 In the industrial Haber process, N2 is reduced to NH3 by H2 at high temperature and pressure with an iron oxide catalyst N2 3H2 (from CH4) 400-500 oC 200 Atm FeO catalyst The The reaction is exothermic at standard temperature & pressure, but has a very high activation energy (420 kJ/mol) in the absence of a catalyst. 2 NH3 3 The roots of leguminous plants have nodules that contain N2-fixing bacteria Rod-like Rod-like bacteria (“bacteroids”) containing the enzyme nitrogenase live symbiotically inside the nodule cells. bacteroids bacteroids Nitrogenase is very sensitive to O2. It is protected in the nodules by leghemoglobin, a heme protein with a strong affinity for O2. Leghemoglobin is produced by the plant, but carries O2 for reduction by the bacterial respiratory chain, keeping the O2 concentration low. 4 (This electron micrograph is colorized artificially.) 2 µm nodule cell nodule nucleus Nitrogenase from Azobacter vinelandii has iron-sulfur and iron-molybdenum centers Azotobacter are free-living, aerobic soil bacteria. Mg ADP (2) Fe protein 4Fe:4S cluster 8Fe:7S cluster Mo-Fe-S Mo-Fe-S cluster (Mo:7Fe:9S) FeMo protein The two ATP-binding sites are structurally homologous to G-proteins. 1n2c.pdb 5 The Fe-Mo cofactor contains homocitrate The mechanism of the N2The fixation reaction is not known. cysteine residue of the protein Fe S Unidentified central atom (probably N inserted from N2) 1N1M.pdb Mo I. Dance, Chem. Commun. (2003) 324-325. I don’t expect you to remember this structure. histidine residue of the protein homocitrate (3-hydroxy-3-carboxyadipic acid) 6 Nitrogenase uses ~16 ATP to reduce N2 to 2 NH4+. It simultaneously reduces 2 H+ to H2 4 CO2 + 4 acetyl-CoA 4 CoA-SH + 4 pyruvate 8 e- ATP is used to transfer electrons one at a time from ferredoxin to the Fe protein. ~16 ADP + 16 Pi 8 ferredoxin (oxidized) 8 e- 8 eFe-Mo protein (oxidized) The Fe-Mo protein reduces N2 and releases H2. ~16 ATP 8 Fe protein (oxidized) 8 Fe protein (reduced) The Fe protein transfers electrons one at a time to the Fe-Mo protein. 8 ferredoxin (reduced) 8 e- 2 NH4+ Fe-Mo protein (reduced) N2 + 8 H + H2 2 H+ 7 Ingested proteins are hydrolyzed by proteases in the stomach (pepsin) and intestine (trypsin & chymotrypsin) Trypsin and chymotrypsin are synthesized in the pancreas as inactive pro-enzymes (zymogens) and are bound by protein inhibitors. Ca Ca proteases in the intestine bovine trypsinogen (cyan & blue) with pancreatic trypsin inhibitor (red) 1tgs.pdb bovine trypsin 2ptn.pdb The same mechanisms are used to regulate other proteases, including the enzymes involved in blood coagulation. Trypsin and chymotrypsin have similar structures and catalytic mechanisms, but different specificities Chymotrypsin hydrolyzes next to the carboxyl group of Phe, Tyr or Trp. Trypsin hydrolyzes next to Arg or Lys The active site has a “catalytic triad”of Asp, His & Ser residues. 2ptn.pdb 1chg.pdb R-CO-NH ... + HO-CH2 ... protein substrate enzyme R-CO-O-CH2 ... ester intermediate H2O + H2N- ... R-CO2H + HO-CH2 ... The first step in catabolism of most amino acids is transamination CO2+ H3N-C-H R amino acid CO2C=O CO2C=O CH2 CH2 CO2+ H3N C H CH2 CH2 CO2- CO2- α-ketoglutarate R α-keto acid glutamate The main function of transamination is to funnel amino groups into a small number of amino acids, particularly Glu, Asp & Ala. Some amino transferases (“transaminases”) are specific for α-ketoglutarate and Glu; others use oxaloacetate and Asp. 10 Transaminases use pyridoxal phosphate as a prosthetic group Enz NH + Lys NH2 + O=CH HO Enz HO HO CH3 Pyridoxal phosphate forms a Schiff-base (aldimine) bond to a lysine residue of the enzyme. This reaction is readily reversible. CH2OH CH2O- P NH NH + Lys N=CH H2O CH3 pyridoxal phosphate + H3NCH2 CH2O- P H2O CH2O- P + CH3 pyridoxamine phosphate NH + Cl - HOCH2 HO CH3 pyridoxine hydrochloride (vitamin B6) 11 Pyridoxal phosphate transfers the amino group by shuttling between aldehyde and amine forms H amino acid 1 - O2C - O2C C R1 NH3+ pyridoxal phosphate CH3 CH2O- P (on enzyme) N H+ H amino acid 2 - O2C C R2 NH3+ α-keto acid 1 CH2NH3+ CH=O HO C R1 O CH2O- P HO CH3 N H+ - O2C C R2 O pyridoxamine pyridox phosphate (on enzyme) α-keto acid 2 Both steps occur with the coenzyme bound non-covalently to the enzyme. This is a classic “ping-pong” enzyme mechanism. 12 amino acid - O2C H H CR NH2 .. H+ CH=O - O2C CH2O- P N H+ H2O H - O2C CR NH C N HO pyridoxal phosphate CH3 Schiff R base CH CH-OH H+ HO CH3 11 The positive charge of the pyridoxine ring facilitates interconversions of Schiff-base intermediates CH2O- P CH2O- P HO N H+ CH3 N H+ H+ α-keto acid - O2C CR O H2O - O2C CH2O- P CH3 N H+ pyridoxamine phosphate + H O2C CH2 CH2NH2 HO - CR N HO CH3 CR N CH CH2O- P N H + Schiff base CH2O- P HO CH3 N H The active site has additional residues that could facilitate proton binding and release Arg 222 Lys 258 Asn 194 Schiff base formed from PLP & 2-methyl-Asp aspartate aminotransferase Asp 222 1ajs.pdb 14 Related enzymes use pyridoxal phosphate to catalyze amino acid racemizations and decarboxylations H H - O2C amino acid - CR NH2 H2O O2C C N R CH=O CH2O- P CH3 H C C R N CH CH2O- P HO H N H+ H N H+ CH2O- P CH3 CO2 CH HO HO Schiff base R CH3 N N H CH amine H C NH2 CH2O- P HO H R H2O CH3 N H+ Schiff base H+ 15 Amino acid decarboxylases generate amines that serve as neurotransmitters CO2+ H3N-C-H - CO2 + H3N-C-H Glu dihydroxy-Phe (DOPA) CH2 - CO2 CO2 5-hydroxyTrp CH2 CH2 OH OH CH2 HO NH CO2 CO2 NH3+ CO2+ H3N-C-H NH3+ CH2 NH3+ CH2 CH2 CH2 CH2 CH2 CO2 - γ-aminobutyrate (GABA) Also: Histidine OH OH dopamine histamine + CO2 CH2 HO NH serotonin 16 Alanine carries amino groups from muscle to the liver for excretion protein muscle glucose blood liver glucose O H2N-C-NH2 urea amino acids NH4+ Glu - CO2 C=O CH3 pyruvate CO2α-keto- +H3N C H Ala glutarate CH3 CO2C=O Glu CH3 pyruvate CO2+ H3N C H Ala CH3 α-ketoglutarate 17 cellular protein ingested protein The amino groups of glutamic acid and glutamine can be released as ammonia in liver mitochondria transaminases CO2+ H3N-C-H amino acids CO2C=O R R α-ketoglutarate α-keto α-keto acids CO2+ H3N C H CH2 CH2 CO2C=O CH2 CH2 - CO2 glutamate dehydrogenase NADH or NADPH + H+ But NH4+ is toxic to the brain, where Glu acts as a neurotransmitter and NH4+ binds to the Glu receptors. Terrestrial organisms must prevent it from accumulating. Glu CO2+ H3N-C-H - CO2 H2O NAD+ or NADP+ NH4+ NH4 CH2 CH2 CONH2 + Gln Gln from muscle & other tissue 18 Ammonia is detoxified and incorporated into many biological molecules through glutamine Glutamine synthetase catalyzes formation of glutamine from glutamate and NH4+ CO2- - CO2 + H3N-C-H + ATP CH2 CO2- H3N-C-H CH2 ADP + NH4+ H3N-C-H Pi CH2 CH2 CH2 - CO2 O CH2 C-O- P O C-NH2 Gln Glu The reaction proceeds through an enzymebound γ-glutamylphosphate intermediate 19 Glutamine serves as a donor of amine groups for synthesis of many other molecules carbamoyl-phosphate In In most terrestrial animals, Gln carries ammonia in the blood to the liver & kidneys, where it is hydrolyzed for excretion as urea. glucosamine-6-P - CO2 + H3N-C-H alanine CH2 glycine CH2 Under conditions of starvation, the liver exports Gln for use in other tissues. O C-NH2 histidine tryptophan urea NH4+ CTP AMP 18 Glutamine synthetase is inhibited by many of the end-products Glu CO2+ H3N-C-H CH2 Gln glutamine synthetase ATP NH4+ ADP Pi Many different Many amino acids and nucleotides bind weakly to the active sites. Each inhibits the enzyme partially. Together, they can shut it down. glucosamine6-P alanine CO2+ H3N-C-H CH2 CH2 CO2- carbamoylphosphate CH2 XXXXXXXX O glycine C-NH2 histidine tryptophan CTP The enzyme also can be inhibited completely by phosphorylation. AMP 19 Bacterial glutamine synthetase has 12 identical subunits that cooperate in the regulation by end-products views of the Salmonella typhimurium enzyme parallel and perpendicular to its 6-fold symmetry axis 2gls.pdb Most human cells have a similar enzyme with 8 subunits. Glial cells in the brain have another Gln synthetase that is constitutively active. Its main role probably is just to remove NH3. 20 E. coli glutamine synthetase also is controlled by adenylylation ATP glutamine synthetase --OH (active) α-ketoglutarate Gln PPi adenylylation glutamine synthetase --O-AMP (inactive) deadenylylation ADP Pi adenyl group O enz O P OCH2 O Adenine O The regulation by Gln and α-ketoglutarate involves similar covalent modifications (uridylylation) of the enzymes that add or remove the adenyl group to glutamine synthetase. 21 ...
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