Abstract
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.
Highlights
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 phosphoglucoisomerasewas studied by phase sensitive two-dimensional 13C-(lH)EXSY NMR spectroscopy at
The shape of the build-up curves of the cross-peaks associated with the 13C2resonances of the a- and 0anomers of both ~-[2-’~C]glucos6e-phosphate and D[2-’3C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between a-D-[2-’3C]glUCOSe 6-phosphate and 8-~-[2-’~C]fructos6e-phosphate
Summary
Phosphate inthe sample containing the active enzyme. Chemicak and sampfe Preparati0n"o-[2-'~C]Glucose(87.5 mM; 99% isotopic purity; Cambridge Isotope Laboratories, Woburn, MA) was converted into D-[2-13C]glUCOSe6-phosphate by a 120-min incubation a t 37 "C in a bicarbonate buffer (25 mM, pH 7.4), equilibrated against a mixtureof 02/C02 (191v, /v) and containing 5 mM MgClp, 100mM ATP, 5mM sodium azide, and 17.5 units/ml yeast hexokinase (EC 2.7.1.1, Boehringer Mannheim). The matrical calculations were performed numerically using the software package EDUMATLAB, Version 1.0 (1988)T.he numerical diagonalization procedures failed for the lowest and highest values of the mixing times, as well in the measurement at 150.869 MHz as in the one a t 125.759 MHz, giving rise to some negative eigenvalues for the matrix @ which makes the calculation of In j3 impossible The reason for this calculation failure is to be sought in the unreliability of the cross-peak and auto-peak volumes associated with the low abundant a-~-(2-’~C]fructo6sephosphate a t very short andvery longmixing times, respectively,due to their poor signal-to-noise ratio. It was pointed out that a weighted average analysis is not applicable to multisite exchanges with widely varying rate constants, because optimum mixing times cannot be determined under such conditions (30)which are met inthe present case
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