Out-and-back 13C–13C scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS

We present here ¹H-detected triple-resonance H/N/C experiments that incorporate CO–CA and CA–CB out-and-back scalar-transfer blocks optimized for robust resonance assignment in biosolids under ultra-fast magic-angle spinning (MAS). The first experiment, (H)(CO)CA(CO)NH, yields ¹H-detected inter-residue correlations, in which we record the chemical shifts of the CA spins in the first indirect dimension while during the scalar-transfer delays the coherences are present only on the longer-lived CO spins. The second experiment, (H)(CA)CB(CA)NH, correlates the side-chain CB chemical shifts with the NH of the same residue. These high sensitivity experiments are demonstrated on both fully-protonated and 100 %-H^N back-protonated perdeuterated microcrystalline samples of Acinetobacter phage 205 (AP205) capsids at 60 kHz MAS.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

Arginine and ornithine catabolism by Clostridium botulinum

Clostridium botulinum 62-A was shown to catabolize l-arginine via citrulline to ornithine, NH(3), and CO(2). The individual enzymes of the dihydrolase system were all demonstrated in extracts of cells, spores, and germinated spores. There was no liberation of urea from l-arginine, so no functional arginase enzyme is present, but there was some transamidinase activity in cell extracts. l-Ornithine was degraded at a significant rate by cells grown in an l-ornithine-supplemented medium; it was partially decarboxylated to putrescine and partially fermented to NH(3), CO(2), volatile acids, and delta-aminovaleric acid. Results from the fermentation of l-ornithine-C(14), -1-C(14), and -2-C(14) demonstrated that essentially all of the CO(2) was derived from carbon 1, and volatile acids from carbons 2 to 5. Assays for the products of l-ornithine-C(14) fermentation revealed that the volatile acids consisted of acetate, propionate, valerate, and butyrate (in order of decreasing concentrations), and that delta-amino-valerate was the primary reduced product. A small amount of citrulline was formed during the fermentation. The carbon and redox balances indicated that l-ornithine is fermented as a single substrate. Preliminary experiments demonstrated that the fermentation of l-ornithine is carried out by cell extracts with the production of volatile acids.     

The periplasmic alpha-carbonic anhydrase activity of Helicobacter pylori is essential for acid acclimation

The role of the periplasmic alpha-carbonic anhydrase (alpha-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since alpha-CA catalyzes the conversion of CO2 to HCO3-, the role of CO2 in periplasmic buffering was studied using an alpha-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that alpha-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the alpha-CA knockout. In the mutant or in the presence of acetazolamide, there was an approximately 3 log10 decrease in acid survival. In acid, absence of alpha-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the alpha-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of alpha-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3- by the periplasmic alpha-CA.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Nitrification of industrial wastewaters with high nitrogen concentration.

Industrial wastes from the production of nitrogen fertilizers, containing about 900 mg N/1, (450 mg NH4-N, 300 mg urea-N and 150 mg NO3-N), showed high activity of I stage nitrifying bacteria. The addition of phosphorus to the wastes increases the intensity of nitrification two-fold and also increase the rate of urea hydrolysis. An attempt was made to obtain a nitrifying activated sludge. CaCO3 and Fe(OH)3 were used as carriers and the culture was aerated with air enriched with CO2. After 90 days an activated sludge was obtained which nitrified an average of 80% NH4-N and urea-N with 4-day aeration time of the wastes. In contrast to the activated sludge, the presence of nitrification phase I and II in biological bed was observed, but the efficiency of the process was considerably low (about 38%).     

Ultraviolet-gas phase and -photocatalytic synthesis from CO and NH3

The major photoproduct obtained on irradiation of gaseous NH3 and CO mixtures is ammonium cyanate; lesser amounts of urea, biurea, biuret semi-carbazide, formamide and cyanide were observed. The formation of the major gas phase photolysis product may be rationalized by the following reaction sequence: (see article). Urea is probably formed from NH4NCO in a thermal reaction while formamide may result from the disproportionation of NH2CO. Photocatalytic syntheses of 14C-urea, -formamide, and -formadehyde are effected by irradiation of 14CO and NH3 in the presence of Vycor, silica gel, or volcanic ash shale surfaces. These syntheses are catalyzed by ultraviolet wavelengths longer than those absorbed by the gaseous reactants. The syntheses are also effected when the surface material is first irradiated in the presence of CO followed by a dark incubation with NH3. Apparently, the initiating step is a light dependent formation of a reactive form of CO on the surface. A discussion is given on the possible contribution of these reactions to the abiotic synthesis of organic nitrogen compounds on Mars, on the primitive Earth and in interstellar space.     

Utilization of carbonate and ammonia-based treatments to eliminate Escherichia coli O157:H7 and Salmonella Typhimurium DT104 from cattle manure

AIMS: The objective of this study was to investigate alkaline treatments of cattle manure to kill coliforms, Escherichia coli O157:H7 and Salmonella Typhimurium DT104 based on their inhibition by carbonate ion and ammonia. METHODS AND RESULTS: Pure cultures of S. Typhimurium DT104 and E. coli O157:H7 strains were treated with sodium carbonate and ammonia to determine threshold inhibitory concentrations. Fresh cattle manure samples were inoculated with the same strains and their survival was determined after addition of sodium hydroxide, ammonium sulphate, sodium carbonate and/or urea. Control of CO and NH3 concentrations in manure by pH adjustment to 9.5 with sodium hydroxide to more than 5 and 30 mmol l-1, respectively, killed more than 106 cells g-1 in 7 days. Addition of sodium carbonate enhanced the killing effect of NaOH by increasing the CO and NH3 concentrations. Addition of 100 mmol l-1 urea, produced high levels of CO and NH3 and decreased all bacterial counts by at least 106 cells g-1 after 7 days. CONCLUSIONS: Reduction of food-borne pathogens in manure can be achieved by a combination of high concentrations of CO and NH3 which are pH-dependent parameters. SIGNIFICANCE AND IMPACT OF STUDY: Addition of urea could provide a simple manure treatment by combining both antimicrobial factors.     

Protein stabilization by urea and guanidine hydrochloride

The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.     

Crystal structure and solution conformation of S,S'-bis(Boc-Cys-Ala-OMe): intramolecular antiparallel beta-sheet conformation of an acyclic cystine peptide

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.     

beta-Alanyl-beta-alanine in cyclic beta-turned peptides.

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(L-Pro-L-Pro-L-Phe-beta-Ala-beta-Ala). The peptide was synthesized by classical solution methods and the cyclization of the free pentapeptide was accomplished in good yields in diluted methylene-chloride solution using N,N-dicyclohexylcarbodiimide. The compound crystallizes in the monoclinic space group P21 from hot water with five solvent molecules. The Pro1-Pro2 peptide bond is cis and the molecular conformation is stabilized by an intramolecular hydrogen bond between the CO group of the beta-Ala5 and the NH of the Phe3 residue. The Pro1-Pro2 segment occupies the relative positions 2 and 3 of a type VIa beta-turn, while the L-phenylalanyl-beta-alanyl-beta-alanine segment is incorporated in a C13-like ring structure. The crystal packing is characterized by a network of 11 intermolecular hydrogen bonds involving all the remaining CO, NH, and the water molecules.     

The effect of urea synthesis on extracellular pH in isolated perfused rat liver.

In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.     

Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins

The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve ¹⁵N and ¹HN resonances. The assignment becomes problematic when there is resonance overlap of ¹⁵N–¹HN cross peaks. For such residues, one cannot unambiguously link the “left” side of the NH root to the “right” side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(i−1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(i−1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i−1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i−1) resonances for overlapping NH roots.     

脲对棒状链霉菌棒酸合成的影响

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。     

Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+, HCN, and CN-

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at alpha-195(His) (replaced by alpha-195(Asn) or alpha-195(Gln)) or at alpha-191(Gln) (replaced by alpha-191(Lys)), were used to probe the interactions of HCN and CN(-), both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C(2)H(2) enhances the rate of CH(4) production from HCN reduction by wild-type nitrogenase. In the absence of C(2)H(2), wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH(4) and NH(3) production increased with increasing NaCN concentration. The NH(3) production rate exceeded the CH(4) production rate up to 5 mM NaCN, at which point they became equal. The "excess NH(3)" likely arises from the two-electron reduction of HCN to CH(2)=NH, some of which is released and hydrolyzed to HCHO plus NH(3). With added C(2)H(2), the rate of CH(4) production increased but only until it equaled that of NH(3) production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C(2)H(2). The increased CH(4) production did not arise from the added C(2)H(2). The lowered total electron flux with C(2)H(2) present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH(2)=NH would be displaced, more CH(2)=NH would undergo the full six-electron reduction, and the rate of CH(4) production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the alpha-subunit of the MoFe protein at positions alpha-191 and alpha-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH(4) production by the alpha-195(Asn), alpha-195(Gln), and alpha-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the alpha-195(Asn) MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH(4) and NH(3) production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the alpha-195(Gln) MoFe protein, the rates of production of both CH(4) and NH(3) were equal at all NaCN concentrations, and total electron flux was hardly affected by changing the NaCN concentration. With the alpha-191(Lys) MoFe protein, the rates of both CH(4) and NH(3) production were very low, but the rate of NH(3) production was higher, and both rates slowly increased with increasing NaCN concentration. A hypothesis, which is based on the varying apparent affinities of the altered MoFe proteins for HCN and CN(-), is advanced to explain the higher rate of NH(3) production versus the rate of CH(4) production and the effect of increasing NaCN concentration on electron flux to products. A new method for CH(3)NH(2) quantification showed that all four MoFe proteins produced CH(3)NH(2). Added CO significantly inhibited both CH(4) and NH(3) production from HCN with all MoFe proteins except for the alpha-191(Lys) MoFe protein, which still manifested its very low rate of NH(3) production but without CH(4) production. All of the MoFe proteins responded differently to the addition of C(2)H(2) to reactions containing NaCN. With the alpha-195(Asn) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production, but the rate of NH(3) production decreased much less. C(2)H(2) also exacerbated the inhibition of electron flux. With the alpha-195(Gln) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production substantially and about equally. C(2)H(2) also eliminated the slight decrease in total electron flux that was caused by NaCN. Added C(2)H(2) hardly affected the alpha-191(Lys) MoFe protein. (ABSTRACT TRUNCA     

Carbonic anhydrases as targets for medicinal chemistry

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc enzymes acting as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate. 16 different alpha-CA isoforms were isolated in mammals, where they play crucial physiological roles. Some of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is secreted in saliva and milk. Three acatalytic forms are also known, the CA related proteins (CARP), CARP VIII, CARP X and CARP XI. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited primarily by two classes of compounds: the metal complexing anions and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO(2)NH(2) (R=aryl; hetaryl; perhaloalkyl; X=nothing, O or NH). Several important physiological and physio-pathological functions are played by CAs present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms represents an attractive goal for the design of inhibitors with biomedical applications. Indeed, CA inhibitors are clinically used as antiglaucoma drugs, some other compounds being developed as antitumour agents/diagnostic tools for tumours, antiobesity agents, anticonvulsants and antimicrobials/antifungals (inhibitors targeting alpha- or beta-CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc.).     

The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ.

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.     

Ring cleavage and degradative pathway of cyanuric acid in bacteria.

The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.     

Continuous production of thienamycin in immobilized cell systems

A novel 2-L bubble column was used to study the continuous, immobilized cell production of thienamycin. Cells of Streptomyces cattleya were immobilized by culturing them in an appropriate growth medium containing 60/80 mesh celite particles. The dilution rate used during the continuous growth phase was 0.2 h(-1). This growth phase was terminated upon the development of heavy cell films (100-500 mum thickness), and the medium was replaced with an appropriate thienamycin production medium. The system was then operated in a batch mode until thienamycin production began. At that time, continuous feeding of the production medium was initiated and the influence of medium composition and dilution rate on CO(2), NH(4), biomass, and thienamycin production investigated. With synthetic production medium, a doubling of the dilution rate from 0.05 to 0.10 h(-1) resulted in a doubling of the thienamycin volumetric productivity. Rates of CO(2) and NH(4) production increased by ca. factors of three and two, respectively. The rate of PO(4) utilization also doubled. When the dilution rate was decreased to 0.05 h(-1), the rates of CO(2) production and PO(4) utilization quickly decreased (i.e., within 3 h). The rates of NH(4) and thienamycin production also decreased but more slowly (i.e., ca. 100 h after the decrease in dilution rate). With complex production medium, the rates of CO(2) production and PO(4) utilization appeared to be a direct function of dilution rate at the dilution rates tested. Thienamycin production in this case was not a function of dilution rate. Comparing the synthetic medium with the complex medium at either dilution rate, the volumetric rate of thienamycin production was higher in the system being fed complex medium. However, the specific activity (units thienamycin/g cell/h) observed with complex medium was lower than that observed with synthetic medium. The higher volumetric productivity observed with complex medium was the result of a high cell loading. The above observations will be discussed in terms of control of thienamycin synthesis and film thickness effects.