A direct NMR method for the measurement of competitive kinetic isotope effects

Journal name:
Nature Chemical Biology
Volume:
6,
Pages:
405–407
Year published:
DOI:
doi:10.1038/nchembio.352
Received
Accepted
Published online

We present a technique that uses 13C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (kcat/Km) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase–catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.

At a glance

Figures

  1. Direct NMR spectroscopic measurement of kinetic isotope effects.
    Figure 1: Direct NMR spectroscopic measurement of kinetic isotope effects.

    (a,b) Use of a 13C nucleus to probe nearby isotopic compositions; nuclei shown with an arrow (↑) are spin active and those without possess no spin; separate resonances arise from either coupling (a) or isotopic chemical shift perturbation (b).

  2. Substrate structures and reaction time-courses for KIE measurements.
    Figure 2: Substrate structures and reaction time-courses for KIE measurements.

    (a) Structure of Neu5Acα2,6LacβSPh. (b) Isotopic labels required for the measurement of 13C- and 18O-KIEs. (ce) 13C NMR spectra of the sialosyl C-3 atom from an approximate 1:2 mixture of singly (w = 13C) and doubly (x = w = 13C) labeled Neu5Acα2,6LacβSPh. (c) Fraction of reaction F1 = 0.00, (d) F1 = 0.63, (e) F1 = 0.79. Dashed lines are a guide to show relative peak height changes. (f) A plot of the change in integrated peak intensity ratios (R/R0) versus F1 for all data from this experiment and the best fit line to equation (1) (KIE = 1.0169 ± 0.0014); the circled points correspond to the spectra shown in d and e. (gi) The 13C NMR spectra from an approximate 1:1 mixture of singly (x = 13C) and doubly (x = 13C, z = 18O) labeled Neu5Acα2,6LacβSPh. Dashed lines are a guide to show relative peak height changes. (g) F1 = 0.00, (h) F1 = 0.65, (i) F1 = 0.77. (j) A plot of R/R0 versus F1 for all data from this experiment and the best fit line to equation (1) (KIE = 1.0388 ± 0.0017); the circled points are from the spectra shown in h and i.

  3. Catalytic pathway and mechanism of sialidases.
    Scheme 1: Catalytic pathway and mechanism of sialidases.

    (a) Hydrolysis of Neu5Acα2,6LacβSPh to give α-sialic acid, which undergoes spontaneous mutarotation to give β-sialic acid. (b) Reaction progress from the Michaelis complex (4S2; skew-boat conformation) through the glycosylation transition state (4H5; half-chair conformation) to the sialosyl enzyme intermediate (2C5; chair conformation).

Compounds

13 compounds View all compounds
  1. Phenyl-N-acetyl-α-neuraminyl-(2→6)-β-D-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
    Compound 1 Phenyl-N-acetyl-α-neuraminyl-(2→6)-β-D-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
  2. N-Acetylmannosamine
    Compound 2 N-Acetylmannosamine
  3. Sodium pyruvate
    Compound 3 Sodium pyruvate
  4. Cytidine 5'-triphosphate
    Compound 4 Cytidine 5'-triphosphate
  5. Phenyl-β-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
    Compound 5 Phenyl-β-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
  6. N-Acetyl-α-neuraminic acid
    Compound 6 N-Acetyl-α-neuraminic acid
  7. N-Acetyl-β-neuraminic acid
    Compound 7 N-Acetyl-β-neuraminic acid
  8. Phenyl-N-acetyl-α-neuraminyl-(2→3)-β-D-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
    Compound 8 Phenyl-N-acetyl-α-neuraminyl-(2→3)-β-D-galactopyranosyl-(1→4)-(1-thio-β-D-glucopyranoside)
  9. CMP-sialic acid
    Compound 9 CMP-sialic acid
  10. [18O2]-Benzoic acid
    Compound 10 [18O2]-Benzoic acid
  11. Phenyl-(4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4)-(1-thio-β-D-glucopyranoside)
    Compound 11 Phenyl-(4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4)-(1-thio-β-D-glucopyranoside)
  12. Phenyl-(2,3-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside)
    Compound 12 Phenyl-(2,3-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside)
  13. Phenyl-(2,3,4-tri-O-acetyl-6-O-[18O0;1]-benzoyl-[6-18O0;1]-β-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside)
    Compound 13 Phenyl-(2,3,4-tri-O-acetyl-6-O-[18O0;1]-benzoyl-[6-18O0;1]-β-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside)

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Author information

Affiliations

  1. Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

    • Jefferson Chan,
    • Andrew R Lewis &
    • Andrew J Bennet
  2. Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada.

    • Michel Gilbert &
    • Marie-France Karwaski

Contributions

J.C., all labeled 2,6-sialoside syntheses and NMR measurements; A.R.L., NMR expertise; M.G., supervisor of enzyme production; M.-F.K., expression and purification of sialyltransferases; A.J.B., project planning and design.

Competing financial interests

The authors declare no competing financial interests.

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  1. Supplementary Text and Figures (516K)

    Supplementary Methods, Supplementary Results, Supplementary Figures 1–4, Supplementary Schemes 1–2 and Supplementary Tables 1–4

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