Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR.
The reversible conversion between D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-[1H] EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates. The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-[2-13C]glucose 6-phosphate and beta-D-[2-13C]fructose 6-phosphate. Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates. The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme. The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D-fructose 6-phosphate both as a substrate and a product, contrary to previous proposals. This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase.
Willem
R
R
Free Universities of Brussels, Belgium.
Biesemans
M
M
Hallenga
K
K
Lippens
G
G
Malaisse-Lagae
F
F
Malaisse
W J
WJ
D002384:Catalysis; D005636:Fructosephosphates / Q000378:metabolism; D019298:Glucose-6-Phosphate; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D006600:Hexosephosphates / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013379:Substrate Specificity
D005636:Fructosephosphates; D005958:Glucosephosphates; D006600:Hexosephosphates; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D007535:Isomerases; D005956:Glucose-6-Phosphate Isomerase