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. The nitrogen cycle. The total amount of nitrogen fixed annually in the biosphere exceeds 1011 kg. Reactions with red arrows occur largely or entirely in anaerobic environments. The redox states of the various nitrogen species are depicted at the bottom of the figure. N2 triple bond contains 945 kJ/mol of bond energy, vs. 351 kJ/mol for CO single bond. The organisms that do this are diazatrophs like Rhizobium. It is carried out by a unique enzyme system, Nitrogenase. It is a complex of two proteins, Fe-protein, a homodimer that has a [4Fe-4S] cluster and two ATP binding sites. The other is the MoFe protein, a heterotetramer (α2β2). Each forms an αβ dimer that associates by 2 fold symmetry. Each dimer has two bound redox centers: (1) a P-cluster which consists of 2 [4Fe-3S] clusters linked an additional FeS. (2) Fe-Mo-cofactor which consists of a [4Fe-3S] cluster & a [Mo-3Fe-3S] cluster bridged by 3 S ions. 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.! 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: 6 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). 7 + Glutamine and Glutamate as key entry points for NH4 Glutamine synthetase Amino acid catabolism + enables toxic NH4 to combine with glutamate to yield glutamine. Transamination reactions collect the amino groups from many different amino acids in the form of Glutamine synthetase L-glutamate. is found in ALL organisms. 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 + 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 • glutamate synthase (NADPH) is an enzyme that catalyzes the chemical reaction • L-glutamine + 2-oxoglutarate + NADPH + H + 2 L-glutamate + NADP+ • Thus, the four substrates of this enzyme are L-glutamine, 2-oxoglutarate (α-ketoglutarate), NADPH, and H+ whereas the two products are L- glutamate and NADP+. • This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH2 group of donors with NAD+ or NADP+ as acceptor. This enzyme participates in glutamate metabolism and nitrogen metabolism. It has 5 cofactors: FAD, Iron, FMN, Sulfur, and Iron-sulfur. • It occurs in bacteria and plants but not animals, and is important as it provides glutamate for the glutamine synthetase reaction. • glutamate synthase (NADH); 2 L-glutamate + NAD+ L-glutamine + + 2-oxoglutarate + NADH + H 13 glutamate synthase (ferredoxin) (EC 1.4.7.1) is an enzyme that catalyzes the chemical reaction 2 L-glutamate + 2 oxidized ferredoxin L-glutamine + 2-oxoglutarate + 2 reduced ferredoxin + 2 H+ Thus, the two substrates of this enzyme are L-glutamate and oxidized ferredoxin, whereas its 4 products are L- glutamine, 2-oxoglutarate, reduced ferredoxin, and H+. This enzyme participates in nitrogen metabolism. It has 5 cofactors: FAD, iron, sulfur, iron-sulfur, and flavoprotein. 14 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. Cumulative feedback regulation of glutamine synthetase Cascade leading to adenylylation (inactivation) of glutamine synthetase. GS is finely regulated by reversible inactivation involving a glutamate-dependent covalent attachment of an adenylyl group to a tyrosyl residue of each 12 subunits. This is catalyzed by an Adenylyltransferase (AT). It catalyses both the adenylation and denadenylation reactions. The adenylation to the 12 indentical subunits does not have to be total and the activity is dependent upon the degree of adenylation. The partially adenylated GS is more sensitive to feedback inhibition than the unadenylated enzyme. The degree of adenylylation is dependent upon over 40 metabolites. AT is a single peptide, 115kD. It is activated by ATP, glutamine and the PII regulatory protein. The activator of deadenylylation is αKG. PII regulatory protein can exist in two forms, uridylylate PII which stimulates deadenylylation and deuridylylated PII which stimulates adenylylation.