Simultaneous observation of the anomerization and reaction rates of enzymatic dehydrogenation of Glucose-6-Phosphate

The hyperpolarization of biological samples using dissolution-DNP has become an attractive method for the monitoring of fast chemical and enzymatic reactions using NMR by taking advantage of 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 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 reproducible 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 towards the β anomer of G6P.