Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes.

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea

The carbon and nitrogen economies of a single cultivar of cowpea (Vigna unguiculata (L.) Walp.cv Caloona) nodulated with either a high H₂-evolving strain (176A27) or a low H₂-evolving strain (CB756) of Rhizobium were compared. The two symbioses did not differ in total dry matter production, seed yield, nitrogen fixed, the spectrum of nitrogenous solutes produced by nodules for export, or the partitioning of net photosynthate within the plant throughout the growth cycle. Detailed examination of the carbon and nitrogen economy of the nodules, however, showed a significant difference between the symbioses. Nodules formed with CB756 lost less CO₂ in respiration compared to the higher H₂-evolving symbioses and this could have been largely responsible for a 36% better economy of carbon use in CB756 nodules during the period of maximum H₂ evolution (48-76 days) and over the whole growth period (20-90 days), a 16% economy. In terms of overall net photosynthate generated by the plant, these economies were equivalent to 5% and 2% of the carbon utilized in the two periods, respectively. From the differences in H₂ evolution and CO₂ production by nodules of the two symbioses, the cost of H₂ evolution was found to be 3.83±0.6 millimoles CO₂/millimoles H₂ for plants grown in sand culture and 1.69 ± 0.48 millimoles CO₂/millimoles H₂ for those in water culture. In both symbioses, the ratio of H₂ evolution to N₂ fixed varied markedly during ontogeny, indicating a significant variation in the relative efficiency and thus metabolic cost of N₂ fixation at different stages during development.     

A comparison of two methods for fitting the integrated Michaelis–Menten equation (Short Communication)

The methods of Atkins & Nimmo (1973) and Fernley (1974) for fitting the integrated Michaelis-Menten equation were compared by using the same sets of simulated experimental data. The method of Fernley (1974) is to be preferred because it gives precise and unbiased estimates of the Michaelis-Menten parameters over a wide range of substrate concentrations. However, the estimates may not be symmetrically distributed, especially at low substrate concentrations.     

Structural components of alginic acid. I. The crystalline structure of poly-beta-D-mannuronic acid. Results of x-ray diffraction and polarized infrared studies

A Structure determination of the naturally occuring marine algal polysaccharide poly-β-D -mannuronic acid is described. The structure consists of 1e → 4e linked D -mannuronic acid chains with the monosaccharide units in the C1 chair conformation. The X-ray fiber diffraction photograph obtained from bundles of fibers prepared from Fucus vesiculosus has been indexed to an orthorhombic unit cell in which a =7.6 Å, b (fiber axis) = 10.4 Å, c = 8.6 Å, the unit cell containing two disaccharide chain segments with space group P2₁2₁2₁. A sheet-like structure involving one intra-chain, one intra-sheet, and one inter-sheet hydrogen bond per monosaccharide is proposed. Features of the chain-packing arrangement are compared with mannan.     

Effect of pO(2) during Growth on the Gaseous Diffusional Properties of Nodules of Cowpea (Vigna unguiculata L. Walp.)

Adaptations of nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium CB 756) to growth in pO₂ ranging from 1 to 80% O₂ (volume/volume) involved both readily reversible mechanisms of adjustment and more stable alterations which together resulted in nodules with widely ranging resistance to diffusion of gases. Those grown in subambient pO₂ (1-5% O₂ were altered such that rapid diffusional adjustment was unable to prevent irreversible loss of nitrogenase on their transfer to higher levels of O₂. Those cultured in 80% had adapted to over-supply of O₂ such that their transfer to lower levels of O₂ limited both nitrogenase and respiratory CO₂ release. There was also some evidence for `protective respiration.' Measurement of diffusional properties based on gas exchange kinetics indicated that gaseous permeability values for nodules from 5 to 40% O₂ were relatively constant around 20 × 10⁻³ millimeters per second, while those for nodules from 1% O₂ were as high as 67.7 × 10⁻³ millimeter per second and from 80% as low as 6.8 × 10⁻³ millimeters per second. Estimates of the thickness of the diffusion barrier ranged from 7.5 micrometers for nodules from 1% O₂ to 71.9 micrometers in those from 80% O₂.     

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O₂ (pO₂; 1–80%, v/v, in N₂) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO₂ tested. In sub-ambient pO₂ (1 and 2.5%) nodules contained specific activities of uricase (urate: O₂ oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O₂ contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O₂, the activities of other enzymes of ureide synthesis were relatively unaffected by pO₂. Short-term effects of pO₂ on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO₂ (1, 2.5 or 5%, v/v in N₂) and¹⁵N₂. Despite a fall in total¹⁵N₂ fixation, ureide synthesis and export was maintained at a high level except in 1% O₂ where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O₂ supply and demand to be maintained over a wide range of pO₂, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance Bureau postgraduate fellowship (to F.D.D.).