Synthesis and NMR spectra of 13C-labeled coenzyme A esters

The synthesis of coenzyme A thioesters of 13C-labeled acetate, propionate, succinate, and methyl malonate is described. The average yields were 94%. The 13C-NMR spectra were determined to provide a reference for the resonance positions of these metabolites. The synthesis of coenzyme thioesters of small-molecular-weight acids labeled with 13C has not been described previously, nor have the resonance positions been previously reported.     

Contribution of exogenous substrates to acetyl coenzyme A: measurement by 13C NMR under non-steady-state conditions

A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply. Two sets of experiments were performed: one in which a metabolic steady state but a non-steady-state 13C distribution was achieved and another in which both metabolism and labeling were not at steady state. In the first group, isolated rat hearts were supplied with [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose. 13C NMR spectra of extracts from hearts perfused under identical conditions for 5 or 30 min were compared. In spite of significant differences in the spectra, the measured contributions of acetate, lactate, and unlabeled sources to acetyl-CoA were the same. In the second set of experiments, the same group of labeled substrates was used in a regional ischemia model in isolated rabbit hearts to show regional differences in substrate utilization under both metabolic and isotopic non steady state. This sensitive probe of substrate selection was also demonstrated in intact hearts where excellent time resolution (3 min) of substrate selection was feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermal unfolding of ribonuclease A. A 13C NMR study.

The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.     

13C NMR studies of bacterial fermentations.

We describe the experimental methods used and the constraints that apply in studies of anaerobic cell metabolism by 13C NMR. We review some of the results of our recent work in this area. Clostridium neopropionicum was shown to ferment ethanol into propionate by the acrylate, non-randomizing pathway. The same metabolic route accounts for 50% of the propionate formed in the complex ecosystem that inhabits the pig's large intestine. The rest is formed via the randomizing succinate pathway. Reductive, hydrogenotrophic acetogenesis was studied in several ecosystems. Although it is usually overshadowed by methanogenesis in the competition for hydrogen, it may become an efficient electron sink when methane biosynthesis is blocked by a specific inhibitor.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

X-ray and 13C solid-state NMR studies of N-benzoyl-phenylalanine.

A crystalline sample of N-benzoyl-DL-phenylalanine 1 and a polycrystalline sample of N-benzoyl-L-phenylalanine 2 were studied using 13C high-resolution solid-state NMR spectroscopy. The X-ray structure of the DL form was established. Sample 1 crystallizes in a monoclinic form with a P21/c space group, a=11.338(1) A, b=9.185(1) A, c=14.096(2) A, beta=107.53(3) degrees, V=1400(3) A3, Z=4 and R=0.053. The principal elements of the 13C chemical shift tensors deltaii for 1 and 2, selectively 13C (99%) labeled at the carboxyl groups were calculated. On the basis of 13C (delta)ii analysis the hydrogen bonding pattern for sample 2 was deduced. Enriched samples were used to establish the intermolecular distance between chemically equivalent nuclei for 1 and spatial proximity in heterogeneous domain for 2, employing the ODESSA pulse sequence. The consistence of the complementary approach covering X-ray data, analysis of the 13C (delta)ii parameters and ODESSA results is revealed.     

13C NMR investigations on Npi-[13C1]carboxymethyl-histidine-119 ribonuclease.

The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.     

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

13C NMR spectroscopy of neolignans

The ¹³C NMR spectra of 15 neolignans of several structural types and two lignans were analyzed and their carbon shifts assigned. The shifts of pyrogallol ether and ethyl phenyl carbinyl ether models were used in this connection. The stereochemistry of a dimeric sideproduct in the preparation of the latter models was determined by ¹³C NMR analysis.     

The thermal unfolding of ribonuclease A A 13C NMR study

The ¹³C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1–7 and between 6 and 70°C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as ‘two-state’, evidence is presented for intermediate unfolding stages at least 10°C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17–24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T₁ and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70°C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others.The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

The biosynthesis of tabtoxinine-beta-lactam. Use of specifically 13C-labeled glucose and 13C NMR spectroscopy to identify its biosynthetic precursors

Tabtoxinine-beta-lactam, an irreversible inhibitor of glutamine synthetase is produced by several pathovars of Pseudomonas syringae. We have examined tabtoxinine-beta-lactam biosynthesis, an important and poorly characterized step in pathogenesis caused by this organism. We have identified the biosynthetic precursors of tabtoxinine-beta-lactam by incorporating 13C from specifically 13C-labeled D-glucose precursors and determining the labeling pattern using 13C NMR spectroscopy. Tabtoxinine-beta-lactam is generated by combining a 4-carbon fragment, a 2-carbon fragment, and a single carbon. The 4-carbon fragment arises from aspartic acid, and the 2-carbon unit is donated from carbons 2 and 3 of pyruvate. The 6-carbon backbone of tabtoxinine-beta-lactam arises from the condensation of fragments from aspartate and pyruvate, probably using reactions analogous to the initial steps in the pathway of lysine biosynthesis.     

Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution.     

Quantitation of model digestive mixtures by 13C NMR.

13C nuclear magnetic resonance (NMR) spectra were obtained at 50.3 and 100.5 MHz for methanolic and aqueous mixtures of sodium taurocholate, 1-monocapryloyl-rac-glycerol, and caprylic acid. Distortionless Enhancement by Polarization Transfer (DEPT) was used to improve spectral sensitivity and resolution, and to generate calibration curves for quantitative determinations of each lipid in methanol. Alternatively, the heights for nonoverlapping peaks in a 13C NMR spectrum acquired with inverse-gated decoupling provide reliable quantitative estimates for each component of the mixture, particularly when the data are obtained in methanol. These experiments also demonstrate the feasibility of detailed NMR structural investigations in model systems for glyceride digestion.     

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.     

13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.