On the Equivalence Point for Ammonium (De)protonation during Its Transport through the AmtB Channel
Structural characterization of the bacterial channel, AmtB, provides a glimpse of how members of its family might control the protonated state of permeant ammonium to allow for its selective passage across the membrane. In a recent study, we employed a combination of simulation techniques that suggested ammonium is deprotonated and reprotonated near dehydrative phenylalanine landmarks (F107 and F31, respectively) during its passage from the periplasm to the cytoplasm. At these landmarks, ammonium is forced to maintain a critical number (~3) of hydrogen bonds, suggesting that the channel controls ammonium (de)protonation by controlling its coordination/hydration. In the work presented here, a free energy-based analysis of ammonium hydration in dilute aqueous solution indicates, explicitly, that at biological pH, the transition from ammonium (NH^sup +^^sub 4^) to ammonia ? (NH^sub 3^) occurs when these species are constrained to donate three hydrogen bonds or less. This result demonstrates the viability of the proposal that AmtB indirectly controls ammonium (de)protonation by directly controlling its hydration.
AmtB exists in the membrane as a homotrimer. Each monomer of this protein forms a channel that passively transports ammonium (NH^sup +^^sub 4^) in the form of its “gas” ammonia (NHa) intermediate across the membranes of bacteria; for conciseness we will henceforth refer to both NH^sup +^^sub 4^ and NH^sub 3^ species, together, as Am. Structural models of AmtB resulting from x-ray diffraction (1,2) have provided initial configurations for a plethora of computational (3-10,13) studies aimed at understanding this channel’s mechanistic aspects and implications for homologous human counterparts.
The center of an AmtB monomer forms a narrow hydrophobic pore (lumen) connecting cytoplasmic and periplasmic vestibules, both accessible to aqueous solution. Diffraction studies revealed an NH^sup +^^sub 4^ binding site in the cytoplasmic vestibule (site Am1 (1,2)) where the cation donates hydrogen bonds to the backbone carbonyl group of A162, the side-chain hydroxyl oxygen of S219, and ~2-3 water molecules (3,5,7). Aromatic groups (F107 and F215) form a floor for site Am1, capping the hydrophobic lumen to help prevent entrance of water from the periplasm (see Fig. 1). These aromatic groups rotate at low free energy cost to allow translocation of Am (3,5,7) under the influence of an electrochemical gradient.
In the presence of AmSO^sub 4^, the x-ray structure (1) displayed three luminal binding sites (Am2, Am3, and Am4-see Fig, 1 A), where Am interacts closely with His residues (H168 and H318). Calculations of the apparent pK^sub a^ of luminal Am (3,10) indicate that these sites may only be occupied by neutral NH^sub 3^. As such, it would appear that the disallowance of permanently charged species in the lumen is the most Am-selective feature of AmtB. An aromatic group (F31) just below site Am4 helps to prevent hydration of the lumen, and provides a low free energy barrier for NH^sub 3^ passage to the cytoplasmic vestibule (Fig. 1, A and B). Just below the lumen, a fifth site (Am5) was revealed by a molecular dynamics (MD) study (3). At this site, calculations of the apparent pK^sub a^ (3) suggest Am must exist in its protonated form, where it donates hydrogen bonds to a carboxyl oxygen of D313, the hydroxyl oxygen of S263, and surrounding water (Fig. 1, A and B),
Combining knowledge of experimental and computational results (1-3,10), it appears that AmtB deprotonates NH^sup +^^sub 4^ between sites Am 1-2, and reprotonates NH^sub 3^ between sites Am4-5 to allow Am flux toward the cytoplasm. However, it is difficult to determine, experimentally, how the channel controls these (de)protonation events. Computational studies, though they should help clarify the (de)protonation mechanism, have proposed disparate explanations (3-5,7). Lin et al. (5) and Nygaard et al. (7) both proposed that a highly conserved Asp residue (D160), whose mutation is known to destroy AmtB’s transport capability (11), plays a key role in NH^sup +^^sub 4^ deprotonation. Lin et al. (5) observed that water forms a hydrogen bonded network between NH^sup +^^sub 4^ at Am1 and the carboxylate of D160. This led them to suggest that the charged carboxylate drives deprotonation at site Am1, and accepts a proton donated by NH^sup +^^sub 4^ using hydronium as an intermediate. On the other hand, Nygaard et al. (7) proposed that deprotonation occurs near site Am2, after NH^sup +^^sub 4^ moves from Am1 across the stacked (F107/F215) aromatic moieties. In this configuration, it was suggested that NH^sup +^^sub 4^ donates a proton to D160 via the backbone carbonyl group of A162 and the amide N-H of G163 using an imidic acid mechanism.
Luzhkov et al. (10) presented results that would suggest that D160 does not function as a proton acceptor. Rather, their calculations showed that the apparent pKa of D160’s carboxylate is downshifted (from its standard value of ~3.9) by 0.3-5.1 units when site Ami is unoccupied. When NH^sup +^^sub 4^ occupies Am1, the apparent pK^sub a^ of D160 shifts even further downward by 9.2 units, making its protonation effectively impossible. Our own results (3), as well as those of Luzhkov et al., showed that D160 is engaged in persistent hydrogen bonds with the protein, and that the negative charge of D160 stabilizes Am in its protonated form, shifting its apparent pK^sub a^ upward by ~4 units. Taken together, these results indicate that the importance of D160, as evidenced by mutational studies (11), is more likely due to recruitment of NH^sup +^^sub 4^ from the periplasm and stabilizing its binding at site Am1 rather than accepting a proton as suggested by Lin et al. and Nygaard et al.
Strolling Down Ammonia Avenue: A Review
What I like about Ammonia Avenue and about the Project in general, is that they always manage to surprise and entertain me. If you want music that hasn’t been done before, then it’s waiting for you in Ammonia Avenue.
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Ammonium ion
Definition:
Ammonium ion test measures the amount of ammonium ions in a blood sample.
Alternative Names:
NH4+ test
How the test is performed:
Blood is drawn from a vein, usually from the inside of the elbow or the back of the hand. The puncture site is cleaned with antiseptic. An elastic band is placed around the upper arm to apply pressure and cause the vein to swell with blood.
A needle is inserted into the vein, and the blood is collected in an air-tight vial or a syringe. During the procedure, the band is removed to restore circulation. Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding.
In infants or young children:
The area is cleansed with antiseptic and punctured with a sharp needle or a lancet. The blood may be collected in a pipette (small glass tube), on a slide, onto a test strip, or into a small container. A bandage may be applied to the puncture site if there is any bleeding.
How to prepare for the test:
Fast for 8 - 12 hours. The health care provider may advise you to withhold drugs that may affect test results.
Drugs that can interfere with the test include thiazide or loop diuretics, barbiturates, acetazolamide, neomycin, and oral kanamycin. Consult the health care provider before this test if you are taking any of these medications.
How the test will feel:
When the needle is inserted to draw blood, you may feel moderate pain, or only a prick or stinging sensation. Afterward, there may be some throbbing.
Why the test is performed:
This test may be performed when a condition that may cause toxic accumulation of ammonia is present or suspected.
Ammonia (NH4+) is produced by cells throughout the body, especially the intestines, liver, and kidneys. In the kidneys, ammonia plays a minor role in the acid/base balance, but is otherwise a metabolic waste product (primarily the result of protein metabolism ).
Most of the ammonia produced in the body is used by the liver in the production of urea. Urea is also a waste product but is much less toxic than ammonia.
Ammonia is especially toxic to the brain and can cause confusion, lethargy, and sometimes coma.
Mechanism of Ammonia Transport by Amt/MEP/Rh: Structure of AmtB at 1.35 Ã…
The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times. Two structurally similar halves span the membrane with opposite polarity. Structures with and without ammonia or methyl ammonia show a vestibule that recruits NH 4+/NH3, a binding site for NH 4+, and a 20 angstrom–long hydrophobic channel that lowers the NH 4+ pKa to below 6 and conducts NH3. Favorable interactions for NH3 are seen within the channel and use conserved histidines. Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3.
Department of Biochemistry and Biophysics, S412C Genentech Hall, University of California–San Francisco, 600 16th Street, San Francisco, CA 94143–2240, USA
Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus
Ammonium transporters (Amts) are integral membrane proteins found in all kingdoms of life that fulfill an essential function in the uptake of reduced nitrogen for biosynthetic purposes. Amt-1 is one of three Amts encoded in the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus. The crystal structure of Amt-1 shows a compact trimer with 11 transmembrane helices per monomer and a central channel for substrate conduction in each monomer, similar to the known crystal structure of AmtB from Escherichia coli. Xenon derivatization has been used to identify apolar regions of Amt-1, emphasizing not only the hydrophobicity of the substrate channel but also the unexpected presence of extensive internal cavities that should be detrimental for protein stability. The substrates ammonium and methylammonium have been used for cocrystallization experiments with Amt-1, but the identification of binding sites that are distinct from water positions is not unambiguous. The well ordered cytoplasmic C terminus of the protein in the Amt-1 structure has allowed for the construction of a docking model between Amt-1 and a homology model for its physiological interaction partner, the PII protein GlnB-1. In this model, GlnB-1 binds tightly to the cytoplasmic face of the transporter, effectively blocking conduction through the three individual substrate channels.
Ammonium bicarbonate - China’s bedrock
For the past 40 years, ammonium bicarbonate (ABC) has been a mainstay of Chinese agriculture. Today, however, its former dominant position is being eroded by urea. In this article, Fan Xiushan, Xu Xiucheng and Duan Ping of the Zhengzhou University of Technology look at ammonium bicarbonate’s history and properties, and the future of the industry in China.
China began production of ammonium bicarbonate in 1958. When aqueous ammonia was used to remove carbon dioxide from dissolved solutions to be used for ammonia synthesis, it was discovered that the resultant reaction product
Ammonium bicarbonate
Ammonium Bicarbonate also called bicarbonate of ammonia, ammonium hydrogen carbonate, hartshorn, or powdered baking ammonia is the bicarbonate salt of ammonia.
Ammonium bicarbonate is formed as shown above and also by passing carbon dioxide through a solution of the normal compound, when it is deposited as a white powder, which has no smell and is only slightly soluble in water. The aqueous solution of this salt liberates carbon dioxide on exposure to air or on heating, and becomes alkaline in reaction. The aqueous solutions of all the carbonates when boiled undergo decomposition with liberation of ammonia and of carbon dioxide:
NH4HCO3 → NH3 + H2O + CO2
Properties
At room temperature Ammonium bicarbonate is a white, crystalline powder with a slight odour of ammonia that can dissolve in water to give a mildly alkaline solution. It is however insoluble in acetone and alcohols. Ammonium bicarbonate decomposes at 36 to 60 °C into ammonia, carbon dioxide and water vapor in an endothermic process (as it is with many ammonium salts) and so causes a drop in the temperature of the water. When reacted with acids carbon dioxide is produced, while reactions with alkalis give ammonia.
Uses
Ammonium bicarbonate was used in the food industry as a raising agent (e.g. for gingerbread, Chinese Youtiao) before the introduction of baking soda. This compound is used as a component in the production of fire-extinguishing compounds, pharmaceuticals, dyes, pigments and it is also a basic fertilizer being a source of ammonia. Ammonium bicarbonate is still widely used in the plastic and rubber industry, in the manifacture of ceramics, in chrome leather tanning and for the synthesis of catalysts.lll
Safety
Ammonium bicarbonate is irritant to the skin, eyes and respiratory system.
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Ammonium acetate
Ammonium acetate is the salt of ammonia and acetic acid.
Properties
It is highly hygroscopic. It decomposes easily at high temperatures into acetamide. It melts at 114 C.
Reactions
Ammonium acetate is useful in the Kovengel reaction. It is often used with acetic acid to create an acidic buffer system.
Synthesis of Ammonium Acetate
Ammnonium acetate can be obtained easily by the reaction with of acetic acid with ammonia.
CH3COOH + NH3 → CH3COONH4
At home it can be made by reacting ammonium hydroxide with dilute acetic acid and evaporating the water. As long as the heat applied is kept at a minimum, the substance would not decompose. Applying a vacuum would improve removal of the water. It should must then be dried.
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