ChemInform Abstract: GEMINAL AND VICINAL (13)C‐(13)C COUPLING CONSTANTS OF 85 PERCENT (13)C‐ENRICHED AMINO ACIDS

Tran-Dinh, S.; Fermandjian, Serge; Sala, E.; Mermet-Bouvier, R.; Fromageot, P.

doi:10.1002/chin.197521074

Cited by 2 publications

(2 citation statements)

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“…70 Hz) is substantially larger than all of the phenylalanine aromatic 'Jcc couplings (ca. 55 Hz) (Tran-Dinh et al, 1975). As expected, in IH-l3C spectra of both IIIG1' (Fig.…”

Section: Identification Of Histidine Imidazole 'H and I3c Spin Systemssupporting

confidence: 85%

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

1993

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IIIG'C is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoeno1pyruvate:glycose phosphotransferase system from Escherichia coli. The 'H, "N, and I3C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGLC have been assigned using two-dimensional IH-"N and 'H-I3C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional I3C-I3C-'H correlation spectroscopy via J,, coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly '5N/'3C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The "N and 13C chemical shifts were used t o determine that His-75 exists predominantly in the NE'-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIG'C, and that His-90 exists primarily in the N6'-H state in the unphosphorylated protein. Upon phosphorylation of the NE* nitrogen of His-90, the N*' nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 'H, "N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK, values for both His-75 and His-90 in IIIG'' and His-75 in phospho-IIIG'C are less than 5.0, and that the pK, value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIG", and implications for proposed mechanisms of phosphoryl transfer are discussed.

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“…70 Hz) is substantially larger than all of the phenylalanine aromatic 'Jcc couplings (ca. 55 Hz) (Tran-Dinh et al, 1975). As expected, in IH-l3C spectra of both IIIG1' (Fig.…”

Section: Identification Of Histidine Imidazole 'H and I3c Spin Systemssupporting

confidence: 85%

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

1993

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“…Fig. 2 shows two typical 125.7-MHz "C-NMR spectra of glutamate obtained from perchloric acid extracts of yeast cells incubated with 100% enriched 12-"Clacetate and 100% enriched [1-'3C]glucose for 2 h. The principle for interpreting "C-NMR spectra of 'T-enriched compounds has been published elsewhere [23,241. The '?C-reso- nance signal of glutamate C4 consists of a singlet (S4) and a doublet (D4), the intensities of which are proportional to (P, + P, + P, + P,) and (P, + P, + P, + P,)/2, respectively.…”

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confidence: 99%

Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by ¹³C‐NMR Spectroscopy

Tran-Dinh¹,

Beganton²,

Nguyen

et al. 1996

European Journal of Biochemistry

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A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2-'3C]acetyl-CoA is not 100% and the total glutamate concentration remains constant or varies during incubation.A comparative study of [ l-13C]glucose metabolism was subsequently carried out using Saccharumyces cerevisiae cells from two different strains (ATCC-9763 and NCYC-239) by 'Y-NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [l-'3C]glucose as a carbon source, the "C-NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-"C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC-239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5 %/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.

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ChemInform Abstract: GEMINAL AND VICINAL (13)C‐(13)C COUPLING CONSTANTS OF 85 PERCENT (13)C‐ENRICHED AMINO ACIDS

Cited by 2 publications

References 1 publication

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by ¹³C‐NMR Spectroscopy

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ChemInform Abstract: GEMINAL AND VICINAL (13)C‐(13)C COUPLING CONSTANTS OF 85 PERCENT (13)C‐ENRICHED AMINO ACIDS

Cited by 2 publications

References 1 publication

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated IIIGlc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated IIIGlc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by 13C‐NMR Spectroscopy

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Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by ¹³C‐NMR Spectroscopy