Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with ¹³C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by ¹³C-NMR spectroscopy. [2-¹³C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-¹³C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate caboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates. Received: November 26, 1997 / Accepted: May 11, 1998     

The 18O isotope effect in 13C nuclear magnetic resonance spectroscopy: mechanistic studies on asparaginase from Escherichia coli

The mechanism of the enzyme asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli was examined using 13C NMR spectroscopy. The pH-dependent oxygen exchange reactions between water and aspartic acid were followed by use of the 18O isotope-induced shift of the resonance positions of directly bonded 13C nuclei. Both L-1- and L-1,4-[13C]aspartic acid were used in experiments with previously 18O-labeled aspartic acid, or in experiments involving the use of 18O-labeled solvent water. Asparaginase catalyzes a relatively efficient exchange between the oxygens of water and those on one carboxyl group of aspartic acid. Exchange at C-4 occurs rapidly but, within experimental error, no exchange at C-1 could be detected. These and related experiments involving the position of 18O incorporation during hydrolysis of aspartic acid beta-methyl ester are all consistent with possible acyl-enzyme mechanisms involving C-4, but do not support a free aspartic acid anhydride mechanism.     

Pyruvate metabolism in Halobacterium salinarium studied by intracellular 13C nuclear magnetic resonance spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to study the metabolism of [2-13C]pyruvate in intact cells of Halobacterium salinarium. The spectra of these cells show that pyruvate is reduced to lactic acid and transaminated to alanine. The intensity of C-2 lactate is higher under anaerobic conditions than under aerobic conditions. When cells are grown in the absence of glucose, the level of C-2 lactate intensity is lower. In extracts of these cells, the level of NADH-dependent lactate dehydrogenase activity is lower than that of cells grown in the presence of glucose. A C-5 glutamate resonance suggests the entry of pyruvate into the tricarboxylic acid cycle through acetyl-coenzyme A. In addition, the label is also observed at C-3 and C-4 of glutamate, signifying a pyruvate carboxylase-type reaction and scrambling of label at the fumarate-succinate stage plus malic enzyme operation, respectively. Citrate synthase and malic enzyme activity appear to be controlled by the growth conditions of H. salinarium.     

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

In vivo 13C nuclear magnetic resonance spectroscopy using a solenoid transmit/receiver coil

The administration of cadmium (1.25 mg as Cd2+/kg, ip.) to male rats resulted in a significant increase of hepatic and renal ornithine decarboxylase activity. The maximum increase of ornithine decarboxylase activity to about 10-fold of the controls was seen at 4 hr after the administration of cadmium, and the increased enzyme activity was returned to control levels by 12 hr. Cadmium produced somewhat dose-dependently the increase of ornithine decarboxylase activity. The increase of ornithine decarboxylase seen on the administration of cadmium was cancelled by pretreatment of rats with cycloheximide. The treatment of female rats with cadmium also caused the increase of hepatic ornithine decarboxylase activity, but not renal enzyme activity.     

Carbon-13 nuclear magnetic resonance spectroscopy of [2-13C]carboxymethylcytochrome c.

Horse heart cytochrome c has been carboxymethylated under various reaction conditions using [2-13C]bromoacetate. Direct analysis of reaction products using 13C nuclear magnetic resonance spectroscopy shows that the protein can be much more extensively modified than has previously been assumed. The proximity of one carboxymethylmethionine residue to the paramagnetic center of the ferric protein allows it to be distinguished from a more constant carboxymethylmethionine residue on the basis of the chemical shift of its labeled methylene group. Refolding of cytochrome c after alkylation at low pH apparently gives a different configuration of modified methionine residues within the protein compared to that produced by alkylation at neutral pH in the presence of cyanide.     

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.     

13C nuclear magnetic resonance spectra of 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines.

The 13C NMR spectra of all sixteen 1,2-dioctade-cis-enoyl-sn-glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn-glycero-3-phosphorylcholine structure have been reassigned.     

Structural definition of the C5a C terminus by two-dimensional nuclear magnetic resonance spectroscopy

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an α-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel α-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor. Proteins 28:261–267, 1997 © 1997 Wiley-Liss Inc.     

Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

The two Cepsilon-methyl methionine groups in cytochrome c have been chemically enriched (45%) with 13C. Their 13C NMR signals have been monitored in both the oxidized and reduced states and under various solution conditions. Methionine residue 80 showed characteristic chemical shift positions for the reduced Fe(II) and cyano-Fe(III) forms. No signal for methionine 80 was observed in the oxidized Fe(III) form due to the paramagnetic effect of the iron atom to which it is bonded, but the position of the methionine 65 signal was shifted, indicating that it is sensitive to the change of oxidation state. Two well resolved signals were observed at pH 11 for the Fe(III) form but only one was resolved at pH 2, indicating that while methionine 80 is definitely displaced from the iron atom at alkaline pH, it may not be in acid conditions.     

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy.

Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Identification of the oriI-binding site of poliovirus 3C protein by nuclear magnetic resonance spectroscopy

Replication of picornaviral genomes requires recognition of at least three cis-acting replication elements: oriL, oriI, and oriR. Although these elements lack an obvious consensus sequence or structure, they are all recognized by the virus-encoded 3C protein. We have studied the poliovirus 3C-oriI interaction in order to begin to decipher the code of RNA recognition by picornaviral 3C proteins. oriI is a stem-loop structure that serves as the template for uridylylation of the peptide primer VPg by the viral RNA-dependent RNA polymerase. In this report, we have used nuclear magnetic resonance (NMR) techniques to study 3C alone and in complex with two single-stranded RNA oligonucleotides derived from the oriI stem. The (1)H-(15)N spectra of 3C recorded in the presence of these RNAs revealed site-specific chemical shift perturbations. Residues that exhibit significant perturbations are primarily localized in the amino terminus and in a highly conserved loop between residues 81 and 89. In general, the RNA-binding site defined in this study is consistent with predictions based on biochemical and mutagenesis studies. Although some residues implicated in RNA binding by previous studies are perturbed in the 3C-RNA complex reported here, many are unique. These studies provide unique site-specific insight into residues of 3C that interact with RNA and set the stage for detailed structural investigation of the 3C-RNA complex by NMR. Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex.     

Application of 13C nuclear magnetic resonance spectroscopy to molecular structural analyses of antibody molecules

A 13C nuclear magnetic resonance study of a mouse anti-dansyl monoclonal antibody is reported. The antibody molecule was specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. It was possible to observe all the carbonyl carbon resonances of the antibody. Fab and Fc fragments have been obtained from the antibody and used successfully for the assignment of each of the carbonyl resonances to either the Fab or Fc region. It has been shown that the spectrum of the intact antibody is simply those of Fab and Fc superimposed. It has also been shown that site specific assignments of carbonyl resonances can be made by means of a double labeling technique developed by Kainosho and coworkers.     

Structural characterization of ficaprenol-11 by 13C nuclear magnetic resonance.

The location of the internal trans and cis isoprene units in ficaprenol-11 isolated from Ficus elastica was determined by 13C nuclear magnetic resonance. The alignment of the isoprene units was estimated to be in the order: omega-terminal unit, three trans units, six cis units and alpha-terminal cis alcohol unit.