Abstract
The application of two-dimensional (2D) Fourier-transform NMR to the determination of rate constants of complex enzyme-catalyzed reactions in the steady state is described. The yeast phosphoglucose isomerase (EC 5.3.1.9)-catalyzed anomerization of glucose 6-phosphate (Glc-6-P) as well as its isomerization to fructose 6-phosphate (Fru-6-P) was chosen as an example. The 2D technique permitted the simultaneous monitoring of the time course of the anomerization and isomerization steps, from which the various reaction rates were determined. The results obtained in the steady state demonstrate the usefulness of the 2D technique by confirming that the anomerization of Glc-6-P is enzyme catalyzed and that the isomerization of the alpha anomer of Glc-6-P to Fru-6-P is at least 10 times faster than the isomerization of the beta anomer of Glc-6-P. These results are compared with reaction rates obtained by rapid-quench methods and the mechanistic implications are discussed. Extrapolation of these results suggests that the 2D Fourier-transform NMR method should be applicable in intact biological tissues.
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Selected References
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