Nitrogen Entry into Biomass and Formation of Amino Acids: Glutamate's Role, Lecture notes of Biochemistry

Download Nitrogen Entry into Biomass and Formation of Amino Acids: Glutamate's Role and more Lecture notes Biochemistry in PDF only on Docsity! Nitrogen Metabolism- How does Nitrogen Enter the Biomass, and form Amino Acids Overview of the flow of nitrogen in the biosphere. Nitrogen, nitrites and nitrates are acted upon by bacteria (nitrogen fixation) and plants and we assimilate these compounds as protein in our diets. Ammonia incorporation in animals occurs through the actions of glutamate dehydrogenase and glutamine synthetase. Glutamate plays the central role in mammalian nitrogen flow, serving as both a nitrogen donor and nitrogen acceptor. Reduced nitrogen enters the human body as dietary free amino acids, protein, and the ammonia produced by intestinal tract bacteria. A pair of principal enzymes, glutamate dehydrogenase and glutamine synthetase, are found in all organisms and effect the conversion of ammonia into the amino acids glutamate and glutamine, respectively. Amino and amide groups from these 2 substances are freely transferred to other carbon skeletons by transamination and transamidation reactions. Nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons: N2 + 8H+ + 8e- + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi. This reaction is performed exclusively by prokaryotes, by the enzyme complex nitrogenase. The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe-protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3. Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process is generated by photosynthesis, respiration, or fermentation.! 6 Nitrogen fixation by the nitrogenase complex • Reduction of nitrogen to ammonia is exergonic, but costly in terms of amount of ATP required: • The nitrogen triple bond is very stable, with a bond energy of 945 kJ/mol. • The high activation energy is partially overcome by binding and hydrolysis of ATP. The overall reaction is: 7 Role of ATP in nitrogen fixation • The binding of ATP to dinitrogenase reductase, and the subsequent hydrolysis of ATP, result in conformational changes that help to overcome the high activation energy of nitrogen fixation. • Specifically, ATP binding results in lowering the reduction potential (E’°) of the reductase from – 300 mV to –420 mV, which enhances its reducing power. Remember, electrons tend to flow spontaneously from carriers of lower E’° to carriers of higher E’° (think of oxidative phosphorylation). Glutamine and Glutamate as key entry points for NH4 + • Bacteria and plants have glutamate synthase • Glutamate dehydrogenase also provides glutamate Glutamate releases an amino group as NH4+ in the liver using glutamate dehydrogenase. •  This enzyme occurs only in microorganisms, plants and lower animals. •  Ammonia stored in glutamine is transferred to α-KG to form 2 glutamate. The reduction power comes from NADPH or ferredoxin. •  The NADPH-dependent glutamate synthase from Azospirillum brazilense, is a heterotetramer, with α2 & β2 subunits. •  The α subunit FMN and has a [4Fe-3S] cluster on each. •  The β subunit has an FAD site and 2 [4Fe-4S] clusters. •  The rxn is 5 steps that occurs in 3 active sites. [1] The electrons are transferred from NADPH to FAD at active site 1 on the β subunit. [2] Electrons travel from the FADH2 to FMN at site 2 on the α subunit to yield FMNH2 [3] Glutamine is hydrolysed at site 3 to glutamate and NH3 [4] the NH3 moves thru the channel to site 2, where it reacts with α-KG . [5] α-iminoglutarate is reduced by FMNH2 to form glutamate. Glutamate Synthase Overall: NADPH + H* + glutamine + a-ketoglutarate —> 2 glutamate + NADP* Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the liver and NH4+ is liberated in mitochondria by the enzyme glutaminase The large size (MW ca. 620 Kda) and the complex regulation patterns of Glutamine Synthetase (GS) stem from its central role in cellular nitrogen metabolism. It brings nitrogen into metabolism by condensing ammonia with glutamate, with the aid of ATP, to yield glutamine. GS is from S.typhimurium, has Mn+2 bound, and is fully unadenylylated. Feedback Inhibition: Bacterial GS was previously shown to be inhibited by nine endproducts of glutamine metabolism. Each feedback inhibitor were proposed to have a separate site. However, x-ray data show: 1. AMP binds at the ATP substrate site. 2. The inhibiting amino acids Gly, Ala, and Ser bind at the Glu site. 3. Carbamyl-l- phosphate binds overlapping both the Glu and Pi sites. 4. The proximity of carbamyl- phosphate to the amino acid inhibitors hinders their binding to GS. Glutamine Synthetase can be composed of 8, 10, or 12 identical subunits separated into two face-to-face rings. Bacterial GS are dodecamers with 12 active sites between each monomer. Each active site creates a ‘bifunnel’ which is the site of three distinct substrate binding sites: nucleotide, ammonium ion, and amino acid. ATP binds to the top of the bifunnel that opens to the external surface of GS. Glutamate binds at the bottom of the active site. The middle of the bifunnel contains two sites in which divalent cations bind (Mn+2 or Mg+2). One cation binding site is involved in phosphoryl transfer of ATP to glutamate, while the second stabilizes active GS and helps with the binding of glutamate. Hydrogen bonding and hydrophobic interactions hold the two rings of GS together. Each subunit possesses a C-terminus and an N-terminus in its sequence. The C-terminus (helical thong) stabilizes the GS structure by inserting into the hydrophobic region of the subunit across in the other ring. The N-terminus is exposed to the solvent. In addition, the central channel is formed via six four-stranded β-sheets composed of anti-parallel loops from the twelve subunits. 8 zA © ° = < > » = 9 oo © UTP uMP O. @ uridylylation Glutamine synthetase (inactive) a. 5 PP; H20 pote ccce- ala latatalaiateielalalaiatalatalniaialalaiatetaiaainietaleleiateleiantninlalatetelainininialaletataal Glutamine Figure 22-7b Lehninger Principles of Biochemistry, Fifth Edition © 2008 W.H. Freeman and Company The glutamine synthetase reaction is important in several respects. It produces glutamine. In animals, glutamine is the major amino acid found in the circulatory system. Its role there is to carry ammonia to and from various tissues but principally from peripheral tissues to the kidney, where the amide nitrogen is hydrolyzed by the enzyme glutaminase regenerates glutamate and free ammonium ion, which is excreted in the urine. S is present predominantly in the brain, kidneys, and liver. GS in the brain participates in the metabolic regulation of glutamate, the detoxification of brain ammonia, the assimilation of ammonia, recyclization of neurotransmitters, and termination of neurotransmitter signals. GS, in the brain, is found primarily in astrocytes. ] Astrocytes protect neurons against excitotoxicity by taking up excess ammonia and glutamate. Ammonia arising in peripheral tissue is carried in a nonionizable form which has none of the neurotoxic or alkalosis-generating properties of free ammonia. Liver contains both glutamine synthetase and glutaminase but the enzymes are localized in different cellular segments. This ensures that the liver is neither a net producer nor consumer of glutamine. The differences in cellular location of these two enzymes allows the liver to scavenge ammonia that has not been incorporated into urea. The enzymes of the urea cycle are located in the same cells as those that contain glutaminase. The result of the differential distribution of these two hepatic enzymes makes it possible to control ammonia incorporation into either urea or glutamine, the latter leads to excretion of ammonia by the kidney. BIZARROCOMICS.COM Facebook.com/ BizarroComics Don’t walk away when I'm talking to you! Pyridoxal phosphate, the prosthetic group of aminotransferases. (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiff-base (aldimine) linkage to a Lys residue at the active site. The steps in the formation of a Schiff base from a primary amine and a carbonyl group Pyridoxal phosphate, the prosthetic group of aminotransferases. (c) PLP (red) bound to one of the two active sites of the dimeric enzyme aspartate aminotransferase, a typical aminotransferase . - 007 ‘007 Amino acids from ingested —H =O protein Amino acids a-Keto acids coo- coo” =O Hy Hz Hy H, ‘00- r a-Ketoglutarate Glutamate 7 H,N—C—H aN Hy Alanine 7 00" y Glutamine from fom + icf {no musica muscle Hy Hy oo \vH, itamine Alanine Pyruvate Gl ad NHj, urea, or uric acid Transaminases equilibrate amino groups among available a-keto acids. This permits synthesis of non-essential amino acids, using amino groups derived from other amino acids and carbon skeletons synthesized in the cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized.  Although the amino N of one amino acid can be used to synthesize another amino acid, nitrogen must be obtained in the diet as amino acids paras Arginine vane Glutamine Phenylalanine ketone Glutamate |}€—— histidine Tryptophan bodies Proline Tyrosine a7 ™ Isocitrate a-Ketoglutarate Acetoacetyl-CoA / Citric \ crn. - - : ioni Citrate = Succinyl-CoA |= Thsaise cyste ¥ Valine Acetyl-CoA Succinate Oxaloacetate| Fumarate |} Phenylalanine KG, Tyrosine |=}: Malate P t _— Glucose Alanine Cysteine Isoleucine Glycine Leucine Serine Threonine Threonine Asparagine Tryptophan Tryptophan Aspartate Figure 18-15 Lehninger Principles of Biochemistry, Fifth Edition © 2008 W.H. Freeman and Company [_] Glucogenic [_] Ketogenic 32 Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways, and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic. The glutamate dehydrogenase utilizes both nicotinamide nucleotide cofactors; NAD+ in the direction of nitrogen liberation and NADP+ for nitrogen incorporation. In the forward reaction as shown above glutamate dehydrogenase is important in converting free ammonia and alpha-ketoglutarate (a-KG) to glutamate, forming one of the 20 amino acids required for protein synthesis. However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity. In the reverse reaction, glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH. As expected for a branch point enzyme with an important link to energy metabolism, glutamate dehydrogenase is regulated by the cell energy charge. ATP and GTP are positive allosteric effectors of the formation of glutamate, whereas ADP and GDP are positive allosteric effectors of the reverse reaction. Thus, when the level of ATP is high, conversion of glutamate to a-KG and other TCA cycle intermediates is limited; when the cellular energy charge is low, glutamate is converted to ammonia and oxidizable TCA cycle intermediates. The multiple roles of glutamate in nitrogen balance make it a gateway between free ammonia and the amino groups of most amino acids. The equilibrium position of Gdh favors the synthesis of glutamate, but studies show that in vivo it has an ΔG0´=0. All tissues have some capability for synthesis of the non-essential amino acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons into amino acids and other derivatives that contain nitrogen. However, the liver is the major site of nitrogen metabolism in the body. In times of dietary surplus, the potentially toxic nitrogen of amino acids is eliminated via transaminations, deamination, and urea formation; the carbon skeletons are generally conserved as carbohydrate, via gluconeogenesis, or as fatty acid via fatty acid synthesis pathways. In this respect amino acids fall into three categories: glucogenic, ketogenic, or glucogenic and ketogenic. ‘ete scan Col acetylglutamate synthase + HH oO. NHs \ | _&—CH—CHa—CH—COO Glutamate o, NH; So-ctt,—c,—CH—Co0 Glutamate etutamate kinase, @ > ADP Oo jad CHa CHs—CH—COO”_-y-Glutamyl Po phosphate @: glutamate | dehydrogenase ~~, nanp)* oP, Ni Sc—ci,—c#t,CH—Coo W * Glutamate y-semialdehyde nonenzymatic H.c—CHy wd tt-coo ve st Pyrroline-5carboxylate @.: > NADP) pyrroline carboxylate reductase | ie et Sd _cH-c00 Hon oO. sv cme \ I _©—CH—CH2—CH—COO oO N-Acetylglutamate N-Acetylglutamate N-acetylglutamate @ ase, app | pote b-600 N-Acetyl-y-glutamyl phosphate N-acetylglutamate | Sana am dehydrogenase ~+NAD@y* oP N-Acetylglutamate y-semialdehyde . Glutamate | aminotransferase | >acketoglutarate HB crt crte—crn—bat-c 1,0 N-Acetylornithine N-acetylornithinase | BR-cx,—cr,—crt,—CH-Coo Ornithine ornithine | ~ Carbamoyl phosphate carbamoyl- | transferase >Pi LCitrulline argininosuccinate , B- spans synthetase \, ap + pp, Argininosuecinate argininosuccinase > Fumarate 4 Ny ‘CH CHy—CHy—CH-COO [ Arginine HN, 7 HN X-Ray structure of E. coli carbamoyl phosphate synthetase (CPS).! CPS is technically not part of the Urea cycle. It forms carbamyl PO4 which is one of the substrates of the Ornithine transcarbamylase. Eukaryotes have two forms of the enzyme, CPSI is found in the mitochondria matrix, CPSII is cytosolic and part of pyrimidine synthesis. CPSI uses NH3 while CPSII used glutamine as the Nitrogen donor. The CPSI rxn is non-reversible and is the allosteric and rate limiting step of the Urea Cycle. E.coli has only one CPS which is homologous to both enzymes found in eukaryotes. The enzyme exhibits substrate tunneling. The three sites of the rxn are found along a 96A tunnel in the elongate protein. Nitrogen-acquiring reactions in the synthesis of urea Nitrogen- acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. Activation of the ureido oxygen of citrulline in step 1 sets up the addition of aspartate in step 2. Nitrogen-acquiring reactions in the synthesis of urea ; G00 i Nh H.N—C—H WH, ae a AMP --O—C C—N—C—H | TH | CH. PP; |? AMP } A coo" } i (CH,) Aspartate (CH,); COO- : @ [7s @ Ls re aA H; H—C—NH; - Rearrangement leads to - Aspartate addition is - cards addition ofAMP, Far facilitated by displace- oo . Mrulne activating the carbonyl eda ment of AMP, ros CCna ATP oxygen of citrulline. Figure 18-11b Lehninger Principles of Biochemistry, Fifth Edition © 2008 W.H. Freeman and Company