Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase

John N. Barlow, John S. Blanchard

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The K(m) values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for k(cat) were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed. (C) 2000 Elsevier Science Ltd.

Original languageEnglish (US)
Pages (from-to)473-480
Number of pages8
JournalCarbohydrate Research
Volume328
Issue number4
DOIs
StatePublished - Oct 6 2000

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Uridine Diphosphate
Galactose
Intramolecular Transferases
Substrates
Uridine Diphosphate Sugars
High Pressure Liquid Chromatography
3-fluoro-3-deoxygalactose
UDP-galactopyranose mutase
2-deoxy-2-fluorogalactose
Diphosphates
Uridine
Chemical shift
Sugars
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Hot Temperature
Nuclear magnetic resonance
Enzymes

Keywords

  • Fluoro-sugars
  • Tautomerization
  • UDP-(2-deoxy-2-fluoro)-galactose
  • UDP-galactopyranose mutase
  • UDP-sugars
  • Uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Organic Chemistry

Cite this

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title = "Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase",
abstract = "The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The K(m) values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for k(cat) were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed. (C) 2000 Elsevier Science Ltd.",
keywords = "Fluoro-sugars, Tautomerization, UDP-(2-deoxy-2-fluoro)-galactose, UDP-galactopyranose mutase, UDP-sugars, Uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate)",
author = "Barlow, {John N.} and Blanchard, {John S.}",
year = "2000",
month = "10",
day = "6",
doi = "10.1016/S0008-6215(00)00135-X",
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journal = "Carbohydrate Research",
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T1 - Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase

AU - Barlow, John N.

AU - Blanchard, John S.

PY - 2000/10/6

Y1 - 2000/10/6

N2 - The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The K(m) values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for k(cat) were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed. (C) 2000 Elsevier Science Ltd.

AB - The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The K(m) values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for k(cat) were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed. (C) 2000 Elsevier Science Ltd.

KW - Fluoro-sugars

KW - Tautomerization

KW - UDP-(2-deoxy-2-fluoro)-galactose

KW - UDP-galactopyranose mutase

KW - UDP-sugars

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JO - Carbohydrate Research

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