Simultaneous Observation of the Anomerization and Reaction Rates of Enzymatic Dehydrogenation of Glucose-6-Phosphate by Dissolution DNP

The hyperpolarization of biological samples using dissolution dynamic nuclear polarization (dDNP) has become an attractive method for the monitoring of fast chemical and enzymatic reactions using NMR by taking advantage of a large signal increase. This approach is actively developing but still needs key methodological breakthroughs to be used as an analytical method for the monitoring of complex networks of simultaneous metabolic pathways. In this article, we use the deceptively simple example of glucose-6-phosphate (G6P) oxidation reaction by the enzyme G6P dehydrogenase (G6PDH) to discuss some important methodological aspects of dDNP kinetic experiments, such as its robustness and its ability to provide repeatable results as well as the capacity of this time-resolved methodology to test kinetic models and hypotheses and to provide reliable parameter estimates. To illustrate the potential of our approach, we report the first direct and quantitative evidence of selectivity of G6PDH toward the β anomer of G6P.