Chap_22_2010_v1

Chap_22_2010_v1 - Chemical Aspects of Biological Systems...

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Unformatted text preview: Chemical Aspects of Biological Systems Information Pathways (142C/242C) Meetin g Time: Ins tru ctor: TR, 12: 30- 1:4 5 P M, Phelps H all 3 515 Prof. L u c J aeger Co ntact: jaeg er@ chem. ucsb.edu Office: P S BN 464 9A Office Hrs: R , 2 :00 -3:00 PM; by ap pointm ent wade (wg rabow@ chem.ucs b.e du) PSBN 46 38, P hone 5 302 TR (W ad e) : 1 1:00 AM-12:00 AM an d by a ppointment TA: TA Office Hours : http://www.chem.ucsb.edu/coursepages/index.shtml Username: chem_142c and Password: chem_242c Chapter 22: Biosynthesis of amino acids and nucleotides (Complemented with Chapter 18: Amino acid oxidation and the production of urea) => => => => Introduction Overview of nitrogen metabolism Biosynthesis of amino acids Biosynthesis of nucleotides 1 Part 1: overview of nitrogen metabolism A. Fixation of nitrogen (by the nitrogenase complex) B. Incorporation of NH3/ NH4+ through glutamate and glutamine C. Glutamine synthetase: the primary regulatory point in nitrogen metabolism D.The several classes of reactions that play fundamental roles in the biosynthesis of amino acids and nucleotides Part 2: brief overview of amino-acid catabolism in mammals Part 1: overview of nitrogen metabolism Because soluble, biologically active N is scarce in natural environments. Most organisms maintain a strict economy in their use of ammonia, amino acids (aas) and nucleotides (nts). Most important source of nitrogen is air (4/5 of air is N2). Few species can convert atmospheric N2 into useful organic forms of nitrogen (Klebsiella cyano bacteria, Azotobacter, rhizobium (nitrogen fixing bacteria living as symbionts in the roots nodule of leguminous plants)). 2 Nitrogen-fixing nodules from leguminous plants (eg Bird’s-foot trefoil) Bird ’s-foot trefoil in bloom Root of bird ’s-foot trefoil The symbiotic relationship between the plant and the symbiont takes care of both the energy requirements and oxygen lability of the nitrogenase complex, Nitrogen cycle The total amount of nitrogen fixed annually in the biosphere exceeds 1011 kg. A balance is maintained between nitrate and N2. In anaerobic condition, use of NO 3- instead of O2 for generating ATP 1. Fixation reduction 3. denitrification 2. Nitrification (by soil bacteria) Note: In plants and some bacteria,nitrite and nitrate reductases can reduce nitrite and nitrate for incorporation into aas 3 ANaerobic AMMonia OXidation (ANAMMOX) BOX 22-1 Compartments in bacteria! (planctomycetes) Ladderane lipids from the anammoxosome A. Fixation of nitrogen (by the nitrogenase complex) N2 + 3 H2 2 NH3 ΔG’° = - 33.5 kJ/mol exergonic N2: triple bond => 930kJ/mol To brake it, need of high activation energy In industry: 400 °C to 500 °C, high pressure (>100 atms) Bacteria do the same at 0.8 atm and ambient T° N2 + 10 H+ + 8 e- + 16 ATP 2 NH4+ + 16 ADP + 16 Pi + H2 This is performed by the nitrogenase complex Chapter 22 4 Nitrogenase complex Dinitrogenase (tetramer) Dinitrogenase Reductase (Dimer ) Two binding for ATP/ADP and single 4 Fe-4S (reductase ) P cluster (4Fe-4S) ADP/ATP Iron-molybdenum cofactor (1 Mo, 7 Fe, 9 S) with homocitrate (grey) Tetramer (X2x2) => total of 2 Mo, 32 Fe , 30 S ( Dinitrogenase ) 1/2 Acetyl-CoA + 1/2 CO2 Dinitrogenase reductase Dinitrogenase 1/2 CoA + 1/2 Pyruvate Total of 8 é transferred with consumption of 16 ATP 5 FIGURE 22-2 The role of ATP is catalytic rather than thermodynamic Both ATP binding and ATP hydrolysis bring about protein conformational changes that evidently help overcome the high activation energy of nitrogen fixation. ATP binding to reductase shift reduction potential of reductase from -300 to -420 mV 2 ATP are hydrolyzed just before the transfer of one electron to dinitrogenase . Nitrogenase complex is extremely labile in presence of O 2 Half life Dinitrogenase 10 min Reductase 30 sec Free-living bacteria cope with this by: -> Living in anaerobic environments -> Repressing nitrogenase when O 2 is present -> ( azotobacter ) Uncoupling the electron transport from ATP synthesis so that O2 is burned of as rapidly as it enter the cell -> generate heat -> by differentiation: in filamentous fixing cyanobacteria, 1 of every 9 cells differentiate into an heterocyst specialized in nitrogen fixation (thick walls that prevent O 2 from entering) Otherwise bacteria can live as symbionts with plants ( bacteroids bathed in a solution with leghmoglobin) 6 Cell nucleus bacteroids peribacteroid membrane with leghemoglobin B. Incorporation of NH3/ NH4+ through glutamate and glutamine Glu and Gln are present in cells at high concentration. In bacteria, Glu is one of the primary solutes in the cytosol . Its concentration is regulated in response of nitrogen requirements and to maintain an osmotic balance between the cytosol and the external medium. The most important pathway requires two reactions First reaction is catalyzed by glutamine synthetase (found in most organisms) Glutamate + NH4+ + ATP glutamine + ADP + Pi + H+ System for assimilation of NH 4+ by bacteria. In mammals used for converting toxic free NH 4+ into glutamine for transport in blood. Figure 18-8 7 Second reaction is catalyzed by glutamate synthetase (found in bacteria and plants) α-Ketoglutarate + glutamine + NADPH + H+ The net reaction with the previous one is: α-Ketoglutarate + NADPH + NH4++ ATP L-glutamate +NADP+ + ADP + Pi 2 glutamate + NADP+ In animals, there is no glutamate synthase Thus to maintain a high level of glutamate -> Transamination mechanism C. Glutamine synthetase is the primary regulatory point in nitrogen metabolism Subunit structure of glutamine synthetase 12 identical subunits First level of regulation: allosteric regulation of glutamine synthetase Only partial inhibition by each inhibitor but the effect of multiple inhibitors is more than additive -> all eight inhibitors can shut down the enzyme 8 Second level of regulation: covalent modifications of glutamine synthetase Gln Synthetase is inhibited by adenyl transferase (AT) . The activity of adenylate transferase is modulated by PII regulatory protein which is itself regulated by uridyl transferase (UT). Adenylated Tyr residue within Gln synthetase 3rd level of regulation: transcriptional regulation of production of Gln synthetase through PII Uridylated PII mediates the activation of transcription of the gene encoding Gln synthetase through interaction with additional proteins involved in gene regulation. Deuridylated PII mediates a reduction of transcription of the same gene. In summary: the key roles of glutamine -> L-glutamine transports ammonia (NH4+) in the bloodstream by acting as a temporary storage of nitrogen -> L-glutamine can donate the amino group when needed for amino acid biosynthesis (with glutamine amido transferases) -> Excess of L-glutamine is converted into glutamate by glutaminase in mitochondria. Released ammonia can enter urea cycle for excretion. 9 D.The several classes of reactions that play fundamental roles in the biosynthesis of amino acids and nucleotides (1) Transamination reactions and other rearrangements promoted by enzymes containing pyridoxal phosphate (PLP) (Chap. 18) (2) Transfer of one-carbon groups using tetrahydrofolate (H 4 folate ) or Sadenosylmethionine (Ado-Met) as cofactors (Chap. 18) (3) Transfer of amino groups derived from the amide nitrogen of Gln (1) Transamination reactions and other rearrangements promoted by enzymes containing pyridoxal phosphate (Chap. 18) p. 677-679 (PLP) p.677-679 10 A typical aminotransferase or transaminase : aminotransferase or transaminase the aspartate aminotransferase the This reaction is reversible! PLP •All aminotransferases rely on the pyridoxal phosphate cofactor •Typically, α -ketoglutarate accepts amino groups •The linkage is made via nucleophilic attack of the amino group of an active-site lysine side chain. After dehydration, a Schiff base linkage is formed • The Schiff base covalently linked to the enzyme is called internal aldimine internal aldimine Reaction of transamination catalyzed by aminotransferases (or transaminases) The PLP–amino acid Schiff base is in conjugation with the pyridine ring, an electron sink that permits delocalization of an electron pair to avoid formation of an unstable carbanion on the a carbon. p.689-691 11 Transamination A Racemization B Decarboxylation C (2) Transfer of one-carbon groups using tetrahydrofolate or S-adenosylmethionine as cofactors (Chap. 18) H4 folate p.689-691 12 (2) Transfer of one-carbon groups using tetrahydrofolate or S-adenosylmethionine as cofactors (Chap. 18) Conversion of one carbon unit on H4 folate! The carbon group comes from Ser. p.689-691 (2) Transfer of one-carbon groups using tetrahydrofolate or S-adenosylmethionine (adoMet) as cofactors (Chap. 18) methionine adenosyl transferase AdoMet is the preferred cofactor for biological methyl transfers. p.689-691 13 (3) Transfer of amino groups derived from the amide nitrogen of Gln Proposed mechanism for amido-transferases Conserved Cys These reactions are essentially found in nucleotides synthesis X= activating group (e.g. phosphoryl group from ATP ) p.859-860 14 ...
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This note was uploaded on 04/25/2010 for the course CHEM 142c taught by Professor Reich,n during the Spring '08 term at UCSB.

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